| Entomopathogenic Steinernema and Heterorhabditis nematodes (EPNs) are new bioinsecticide. The recovery and development of the infective juveniles (IJs) of these nematodes play an important role in their commercial production. Identification of the genes during the recovery and development provides insight into the understanding of the metabolic pathways of the recovered Us.A cDNA differential expression library from recovered Us of 5. carpocapsae A2A was constructed and the genes of differential expression were identified. A gene named spi-583 which has high homology with serine proteinase inhibitor genes was selected and expressed in protocaryotic (Escherichia coli) expression system.RLM-RACE (RNA ligase mediated Rapid Amplification cDNA End, RLM-RACE) method was used to construct the differential expression library from S. carpocapsae A24 total RNA. 48 differential expressed clones were identified by dot blotting analysis. These differential expressed clones were sequenced and analyzed by bio-information. 34 full-length genes were obtained, including ribosomal protein genes, translation elongation factor 1-alpha genes, serine proteinase inhibitor genes, cell division protein kinase 5 genes etc.A gene (spi-583) was obtained by RT-PCR amplification from the genomic DNA of S. carpocapsae A24. pET expression system was applied to construct correct recombinant E. coli BL21 (DE3) After over 100 passages, the recombinant showed stable plasmid inheritance and consistence. When recombinant E. coli was induced with 1 mmol/L IPTG at the early exponential phase at 25℃, SPI-583 protein was detected as inclusion bodies with the molecular mass of approximately 19.54 kDa. Western blot analysis confirmed the expression of the SPI-583 protein.These results would be helpful not only to explore nematode-bacterium symbiotic mechanism, but also promote the commercial production of these nematodes.
|
PDF Full Text Request