Dynamic Proliferation Of EoNPV And Hemolin Gene Cloning And Expression Analysis In Ectropis Obliqua And Ectropis Grisescens

Ectropis obliqua Prout and Ectropis grisescens Warren are two major types of leaf-eating pests in tea garden, and the evolution relationship between these two sympatric pests is very close. Ectropis obliqua Nuclearpolyhedrosisvirus(EoNPV) is one of the major pathogenic microrganism of E.obliqua, which was widely used for E.obliqua control for many years. EoNPV is a kind of species-specific baculovirus. More recently, an interesting research results suggest that EoNPV could infect E.obliqua efficiently as well as E.grisescens. Therefore study on the degree of variance pathogenicity and the molecular mechanism of immune interactions between E.obliqua and E.grisescens in response to EoNPV seems very necessary. In present study, we chose E.obliqua and E.grisescens as the experiment insect. Frist, we tested the EoNPV dynamic proliferation in the larvae of E.obliqua and E.grisescens after infected with EoNPV. Second, we construct the midgut transcriptome of E.obliqua after EoNPV infected 12 hours and the normal control. Finally, the molecular cloning and expression profiles of hemolin gene in E.obliqua and E.grisescens were analyzed. The main conclusions were as follows:1 We monitored the dynamic proliferation of Eo NPV in the larvae and the midgut of E.obliqua and E.grisescens by performing real-time qPCR, in order to gain a better understanding of the invasion and the replication properties of EoNPV. In an early stage of EoNPV infection(12 h pi), there is a significant difference of the relative numbers of EoNPV copies between E.obliqua and E.grisescens. As the infection progressed after EoNPV infected 96 h, the relative numbers of EoNPV copies in E.obliqua larvae and midgut organization is 14 times and 8 times than in E.grisescens respectively. The results indicated the EoNPV invaded the midgut tissue and larvae of both E.obliqua and E.grisescens, but the viral proliferation in the midgut and the larve of E.grisescens was significantly inhabited by unknown mechanisms.2 Through the next-generation high-throughput techniques, the transcriptome of midgut of E.obliqua after EoNPV infected 12 hours and the normal control was successfully constructed. 25869 unigenes was generated after assembled, with an average length of 717 bp. And 13733(53.1% of the total unigenes) unigenes were annotated to homology sequences of 7 databases(Nr, Nt, Pfam, Swiss-Prot, GO, KOG and KEGG). According to GO analysis, 9246 unigenes were assigned to three main categories(biological process, cellular component and molecular function) 55 second categories, in which 43 unigenes were assigned to immune system process. KOG analysis can classify into the 26 group, in which “general function prediction only” group contains the highest number of unigenes(1278), and some unigenes located in the defense mechanism and signal transduction groups may have putative function related to virus immune. KEGG pathway analysis showed that 198 unigenes were relevant to immune system. A total of 133 differentially expressed genes were detected after comparing the EoNPV infected 12 hours and the normal control, including 80 up-regulated and 53 down-regulated genes respectively.3. Bioinformatics characterization results showed that cDNA full length of hemolin of E.obliqua was 1772 bp(Gen Bank Accession number: KM885983), which contained an intact ORF of 1239 bp and encoded 412 amino acid residues, The putative molecular mass and an isoionic point was 45.8kD and 8.297, respectively. Sequence-similarity between E.obliqua and E.grisescens was 99%, and molecular mass and isoionic point of hemolin gene of E.grisescens was 45.5kD and 8.265 respectively. The deduced amino acid sequence demonstrates it belongs to a typical secretory protein that contains 4 Ig functional conservative areas and 2 N-glycosylation sites. The results of quantitative real-time PCR analysis further revealed a significant up-regulated expression about 36.5 in the larvae that infected EoNPV than the controls in E.obliqua and 25.3 times in the larvae that infected EoNPV than the controls in E.grisescens. The study indicated that the hemolin gene may participate in the immune response to EoNPV in E.obliqua and E.grisescens.