Study On Several Immune And Epigenetic Genes Related To Spodoptera Frugiperda In AcMNPV Sensitive Host

Baculoviridae is a group of double strand DNA viruses that only infects arthropod in nature. Autographa californica nucleopolyhedrovirus (AcMNPV) is the type species of Baculoviridae. Because baculovirus are highly pathogenic to host insects while very safe to non-target organisms, they does not pollute the environment, and the pest does not develop resistance easily, dozens of baculovirus have been successfully used for the prevention and control of agricultural pests. However, baculovirus also have some disadvantages that limit their application. Safe and effective applications of this virus will depend on our understanding of its biology and virus-host interaction. Since the AcMNPV genome sequencing was completed in1994, the function of many essential genes has been elucidated. However, the study on its interactions with host still remains in the molecular biology stage. In fact, baculovirus establish a parasitic relationship with its insect host in the long-term evolutionary process. The interaction between these two organisms occurs in a multiple levels. In the current research, we explored the interaction between baculovirus and its host insect in terms of Toll signalling pathway, DNA methylation and microRNA.Spodptera frugiperda is a susceptible host for AcMNPV, and Sf9cells isolated from S. frugiperda is the most commonly used cell line for AcMNPV research. To investigate the possible innate immune response during baculovirus infection, we cloned full-length cDNAs of three key components of the Toll signal pathway from the S. frugiperda,18-wheeler (18w), Spatzle, and MyD88. S. frugiperda18w (Sf18w) encodes a protein of1,274residues which shares56%and44%sequence identities with18w protein of Bombyx mori and Drosophila melanogaster, respectively. The S. frugiperda Spatzle (SfSpatzle) encodes a protein of253residues, which shares50%and52%sequence identity with Manduca sexta Spz1A and Spz1B. When SfSpatzle was aligned with other insect Spatzles, a conserved proteolytic processing site was found in it. The S. frugiperda MyD88(SfMyD88) has the Death and TIR domains which is conserved among known MyD88and belongs to the DD-superfamily. Acute immune challenges experiments showed that the expression of18w and Spatzle were stimulated in fat body and midgut after injection of Staphylococcus aureus into the Spodoptera litura larvae. Meanwhile, the antimicrobial polypeptide Gloverin was greatly up-regulated in the S. aureus challenged larave. The same increase in Gloverin expression by Sf9cells was also observed when we treated cell with the hemolymph extracted from the S. aureus challenged larvae. These observations indicate Toll-related genes may function in the innate immune response against Gram-positive bacteria in the Spodoptera insects. However, when we tried to overexpress one or two Toll-related proteins in Sf9cells in attempt to stimulate the expression of Gloverin, which is proved to have anti-AcMNPV function, we did not observe the expected result in any combination of treatment. We speculated that Sf9cells may lose some immunity due to long time in vitro culture, and Toll-related signal pathway cannot be activated, thus provide AcMNPV a suitable environment to replicate.In high eukaryotes, DNA methylation acts as an important epigenetic mark that is associated with many important biological processes such as gene repression, X-chromosome inactivation, and genomic imprinting. Epigenetic alteration is also known to be responsible for some human diseases. DNA methylation is catalyzed by DNA methyltransferases (DNMTs). In eukaryotes, DNMTs can be classified into three subfamilies, namely DNMT1, DNMT2, and DNMT3. The DNMT2subfamily proteins exhibit some unique features and have yet undefined biological function(s). Preliminary research in our laboratory indicated that the methylation level of viral DNA changes in a regular way during AcMNPV infection in Sf9cells, the mechanism and importance of which is still unclear to us. In order to study the role of DNA methylation in virus replication, we identified and cloned a DNMT2gene from S. frugiperda (S/DNMT2). It shares moderate to high sequence identity with known DNMT2s.5/DNMT2is found to exist in both nucleus and cytoplasm and has an evident DNA methyltransferase activity in in vitro enzyme assay. We also determined the crystal structure of SfDNMT2in complex with its cofactor SAH. Like human DNMT2, SfDNMT2is composed of a catalytic domain and a target recognition domain (TRD) yet with a well-defined catalytic loop. The bound SAH has extensive interactions with the surrounding residues. The catalytic loop together with TRD forms a narrow channel connecting the cofactor-binding site which allows the binding of a nucleotide base, suggesting that SfDNMT2might employ a catalytic mechanism similar to the classic DNA methyltransferases. These results provide new insights into the function and the catalytic mechanism of DNMT2in insects. In addition, we also cloned DNMT1from Sf9cells. This protein also has high sequence similarities with homologous protein in other insects. Structure analysis showed it has many conserved domains, included the methyltransferase catalytic domain, RFD domain, CXXC domain and two BAH domains. Its function is currently under investigation. MicroRNAs are a kind of small RNAs belonging to post-transcriptional regulators which have many crucial roles in development, carcinogenesis and so on. To explore whether there are microRNA regulation during AcMNPV infection, we sequenced three small RNA libraries prepared from AcMNPV-infected Sf9cells (6h post-infection (p.i), and12h p.i), and a control cell sample. With the aid of Illumina deep-sequencing technology, we identified30novel pri-miRNAs from virus. We also identified several host microRNA, some of them are novel while the others are conserved. Many of the host miRNAs are up-regulated in AcMNPV-infected cells. The target and function of these miRNAs are under further investigation.