Abstract The silkworm Bombyx mori (Lepidoptera: Bombycidae) is a lepidopteran model insect of great economic importance. The parasitoid Exorista sorbillans (Diptera, Tachinidae) is the major pest of B. mori and also a promising candidate for biological control. However, the molecular interactions between hosts and dipteran parasitoids have only partially been studied. Gene expression analysis by reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is indispensable to characterise their interactions. Accurate normalisation of RT-qPCR-based gene expression requires the use of reference genes that are constantly expressed irrespective of experimental conditions. In this study, the expression stability of 13 traditionally used reference genes was estimated by five statistical algorithms (ΔCt, geNorm, Normfinder, BestKeeper, and RefFinder) to determine the best reference genes for gene expression studies in different tissues of B. mori under E . sorbillans parasitism. Specifically, TATA-box-binding protein was the best reference gene in epidermis and testis, while elongation factor 1α was the most stable gene in prothoracic gland and midgut. Elongation factor 1γ , ribosomal protein L3 , actin A1 , ribosomal protein L40 , glyceraldehyde-3-phosphate dehydrogenase and eukaryotic translation initiation factor 4A were the most suitable genes in head, silk gland, fat body, haemolymph, Malpighian tubule and ovary, respectively. Our study offers a set of suitable reference genes for gene expression normalisation in B. mori under the parasitic stress of E . sorbillans , which will benefit the in-depth exploration of host-dipteran parasitoid interactions, and also provide insights for further improvements of B. mori resistance against parasitoids and biocontrol efficacy of dipteran parasitoids.
Abstract An electrochemiluminescent (ECL) method has been developed for the determination of melamine based on the inhibition of luminol ECL. A significant luminol ECL can be found at 1.47 V in the phosphate buffer solution at high pHs and low potential scan rates, this ECL signal can be inhibited obviously by melamine. The decrease of ECL intensity was linearly proportional to the logarithm of melamine concentration in the range of 1–100 ng/mL ( R 2 =0.9911) and with the detection limit of 0.1 ng/mL. The method has been applied successfully to determine melamine in dairy products and melamine tableware, the recoveries were in the range of 98.5%–103.7% and 95.5%–106.0%, respectively. The mechanism of the inhibition effect was also proposed, the active oxygen (O 2 · − ) generated from the electrooxidation of OH − reacted with luminol anion (L · − ) to generate light emission, and the present of melamine can eliminate the active oxygen, which cause the decrease of the ECL intensity.
We have developed a proteomics technology featuring on-line three-dimensional liquid chromatography coupled to tandem mass spectrometry (3D LC-MS/MS). Using 3D LC-MS/MS, the yeast-soluble, urea-solubilized peripheral membrane and SDS-solubilized membrane protein samples collectively yielded 3019 unique yeast protein identifications with an average of 5.5 peptides per protein from the 6300-gene Saccharomyces Genome Database searched with SEQUEST. A single run of the urea-solubilized sample yielded 2255 unique protein identifications, suggesting high peak capacity and resolving power of 3D LC-MS/MS. After precipitation of SDS from the digested membrane protein sample, 3D LC-MS/MS allowed the analysis of membrane proteins. Among 1221 proteins containing two or more predicted transmembrane domains, 495 such proteins were identified. The improved yeast proteome data allowed the mapping of many metabolic pathways and functional categories. The 3D LC-MS/MS technology provides a suitable tool for global proteome discovery.
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