Study On Differentially Expressed Proteins In Posterior Silk Glands Of Fifth Instar Antheraea Pernyi Larvae

Antheraea pernyi is a typical wild silkworm with larval silk glands specific for the synthesis and secretion of silk proteins.When the larva develops to the fifth instar stage, fibroin is synthesized and secreted in abundance and the metabolism and subcellular structure of the PSG undergoes considerable changes.To study the functions of different expression proteins associated with fibrion preparation and synthesis,two-dimensional electrophoresis and MALDI-TOF-MS were used to identify the upregulated proteins in PSG of A.pernyi fifth instar larvae.Real-time quantitative PCR was performed to detect changes of GSTT and RPL8 at RNA transcription level.Sample preparation is the first and important step towards successful two-dimensional gel electrophoresis(2-DE) and identification in proteomics study.Two-step method which separated the soluble and disolube proteins was proved to be more suitable for silk glands protein extraction to avoid the influence of abundant protein on 2-DE.2D clean-up kit was used to provent horizontal streaks.A simple and economic IEF was performed by Bio-lyte for 2-DE.The optimal condition for 2-DE of PSG protein was 30μg;pH 4～7;1000V,3h.By comparing the 2-DE profiles with day 1 of fifth instar,more changes of protein spots were found on day 4.The 2-DE profiles of days 1 and 4 were obtained through the method of IPG strip and about 350 spots were detected on each profile.After analyzing by Imagemaster,23 unique or upregulated spots(above two folds) on day 4 of the fifth instar were selected to be identified by MALDI-TOF-MS.After searching NCBI database,seven proteins which probably involved in the regulation of transcription,translation,and general cell metabolism were identified.GSTT and RPL8 were cloned and its Genbank accession numbers were EU541490 and EU541491.Real-time quantitative PCR was performed on these two genes to determine the changes at RNA transcription level.The result revealed that the changes at RNA expression level were in corresponding with the protein expression level.