Analysis Of Differential Expression Proteins Between Chorion Of Diapause Egg And Non-diapause Egg Of Silkworm, Bombyx Mori

Oogenesis is a critical physiological process for the insect, especially in reproducing and populations maintainance. Oogenesis goes through previtellogenesis, vitellogenesis, choriogenesis, oviposition and fertilization in insect. During the choriogenesis stage, follicular epithelial cells(FCS) in ovarian tissue synthesize and secrete some special structural proteins which are attached to the surface of oocyte and forming a complex extracellular structural layer called eggshell. Eggshell has important physiological roles in mechanically protecting the embryo, maintaining gas exchange, preventing moisture loss, pathogens entry and assisting the sperm entry. Proteins accounts for 96% in the dry matter of insect eggshell. Analysis of the structure and function of eggshell is one of the hotspots of insect biology research.Diapause, an important biological phenomenon in insect, is one of key reasons that insects have the ability to survive in nature for such a long term. Diapause occurs in the egg, larva, pupa or adult stage. As the model organism of Lepidoptera, silkworm(Bombyx mori) is a typically type of egg diapause insect. When the embryo develops to the blastocyst stage, the cell division stops to enter diapause. After several months of winter, diapause eggs develop and hatch in the following spring. This period is inseparable from the protection of the eggshell. Therefore, eggshell plays a particularly crucial role in the completion of silkworm’s entire life cycle. The non-diapause eggs, however, will hatch after a few days in the nature environment without going through winter. Considering the presence of diapause egg special stage, silkworm eggshells are not only different from other insect species, but also exhibit intraspecific differences even in diapause eggs and non-diapause eggs of silkworm.This paper researched and explored the eggshell proteins associated with diapause by comparative proteomic methods. Here, we used silkworm, Bombyx mori, which is a model species for Lepidoptera as research material. Firstly, we observed the surface structure of the eggshell of silkworm by means of Scanning Electorn Microscope. Then, applying two-dimensional electrophoresis/matrix assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS) and comparative proteomics technology, we constructed the protein profiles of silkworm eggshell and screened the differential proteins between diapause and non-diapause eggshell. Bioinformatics were carried out to analyze the structure and function of differential proteins. And then, we used q RT-PCR to detect the expression levels of genes corresponding differential proteins in ovarian tissuse. The main results and conclusions are showed as follows: 1. Surface structure and construction of the protein profiles of eggshellWe used the bivoltine race Dazao P50 eggs as research materials. Under the different conditions(25°C, long light;15°C,darkness), silkworm eggs were incubated and then reared to oviposit diapause eggs and non-diapause eggs separately. Under the scanning electorn microscope, we observed prominent differences between the surface structure of diapause eggshell and non-diapause eggshell.Then, their eggshell protein samples were extracted. Applying two-dimensional electrophoresis(2-DE) and matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS), 28 protein spots with high abundance in 2-DE maps were identified, 14 protein spots respectively, including chorion class A protein、chorion class B protein、chorion class CA protein、chorion class CB protein and chorion class high-cysteine protein. Finally, the protein profiles of above tissues were constructed. 2. The identification and function analysis of the differential proteinsComparative proteomics analysis was carried out to compare the 2-DE maps of the eggshell proteins and five different spots were displayed. By MALDI-TOF-MS, they were respectively identified as chorion class high-cysteine A12 protein、chorion class B protein L12、chorion class A protein L11、chorion class B protein M2410 and chorion class CA protein ERA.2. However, the most significant difference protein spot was identified as chorion class high-cysteine A12 protein. Then we analyzed its hydrophobicity, disulfide bonds, secondary structure and functions by bioinformatics analysis methods, the results were coincided with the characteristics of Hc proteins. These results provide some references for researching silkworm diapause. 3. The structure analysis and q RT-PCR verification of the genesTo validate the results of 2-DE, we found out the genes expressing differential proteins identified on NCBI and analysed their structure. And quantitative real-time PCR(q RT-PCR) were then used to detect the transcription levels of the genes in ovarian tissues during pupal stage. The results revealed that genes expression levels of the chorion class high-cysteine A12 proteins markedly down-regulated in non- diapause eggs. High-cysteine proteins, being easy to form disulfide bonds which are the main force in stabilizing the organization of the eggshell, are distributed the whole eggshell and are closely related to the firmness and waterproofing of the eggshell. Thus, it’s likely that the higher expression level of Hc proteins in diapause eggs is to form a more highly tough, rigid and relatively waterproof outer layer on the surface of the embryo. During the cold, dry winter months, the outer layer can resist the adverse environment, ensuring the embryos undergo diapause period safe and sound.This study has also shown that the test system, 2-DE and matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS),are proved to be a rather useful indicating system for determining comparative proteins between diapause eggs and non-diapause eggs.