Nosema bombycis contains functional aquaporins (NbAQPs), which are key targets for exploring the mechanism of N. bombycis infection; however, the regulation of these NbAQPs remains unknown. The two highly conserved asparagine-proline-alanine sequences (NPA motifs) play important roles in AQP biogenesis. As part of this study, we constructed a series of NbAQP mutants (NbAQP_NPA1, NbAQP_NPA2, and NbAQP_NPA1,2) and expressed them in BmN cells. The results showed that mutations in either NPA motif or in both NPA motifs did not affect NbAQP expression in vitro. After expression in Xenopus laevis oocytes, those injected with wild-type NbAQP rapidly expanded, whereas oocytes injected with NbAQP_NPAs did not significantly change in size. The associated water permeability (pf) of NbAQP_NPAs was significantly reduced five-six times compared to that of wild-type NbAQP. These results indicated that NPA motifs are necessary for the water channel function of AQPs in N. bombycis. The present study shows for the first time that the NbAQP NPA motif has an impact on the water permeability of aquaporin in N. bombycis, thereby providing a platform for further research into the mechanisms underlying the regulation of NbAQP expression.
Salt-induced self-aggregation of gold nanoparticles (AuNP) carrying the ssDNA probes can be prevented specifically by complementary DNA. Loop-mediated isothermal amplification (LAMP) can amplify DNA rapidly. In the present study, we established a LAMP-AuNP method for detection of the microsporidium Nosema bombycis by combining the two techniques. The DNA templates of microsporidia were extracted by FTA card. The amplified product can be visually detected via hybridization at 63°C for 5 min with a ssDNA-labelled nanogold probe, followed by salt-induced AuNP aggregation. The LAMP-AuNP assay needs approximately 65 min. This method can detect 10 spores/ml and is specific for N. bombycis. When it was used to detect pebrine disease of the silkworm eggs, the maximum sample size for silkworm eggs was 700, in which only one egg was infected with N. bombycis. The new LAMP-AuNP is a simple, sensitive and specific detection assay for N. bombycis.
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