| Aphids are important agroforestry pests and sucking plant sap with their special piercing-sucking mouthparts. But some of them are significant resource insects such as chinese gall aphids which may induce galls on Rhus. Aphid saliva, secreted by salivary gland, is the media of insect-plant interaction, which plays a crucial role in aphid-host plant interactions. Analyzing the protein components of aphid saliva and salivary gland will helps to reveal the mechanism of aphid-plant interaction and provides scientific evidence for pests control and the utilization of useful species. The thesis mainly analyzes the secreted proteins of Schlechtendalia chinensis. Proteome methods were used to detect and identify the saliva and salivary gland proteins. The saliva collected by a double layer ParafilmTM was concentrated by ultrafiltration membrane. The dissected salivary gland was used to extraction proteins by ultrasonication associated lysis byffer. Both the collected saliva and salivary gland proteins were detected by Bradford, SDS-PAGE and LC-MS/MS analysis. All data collected by MS were compared against the Acyrthosiphon pisum protein database by Mascot software. The results of our protein identification may provide beneficial evidences of the aphid-plant interaction and the aphid gall formation. The main results are as follows:(1) The extraction method of the saliva proteinsUsing the double layer of Parafilm, we collected the saliva of the S. chinensis. Then we used concentration, vacuum freezing and ultrafiltration membrane to concentrate the saliva, respectively. The results showed that the ultrafiltration membrane is the best way to extraction the saliva proteins, and it would be uesed in the following experiments.(2) The collection method of the salivary glandsFor doing this experiment, enough salivary glands are needed. Dissected undering the disscting microscope, we practiced a lot to make sure that we can dissect out the salivary gland quickly and exactly. Using the lysis buffer assisted with the sonication to homogenized the salivary glands. We quantified and separated the proteins of the salivary glands by using the ultraviolet spectrophotometer/Bradford/SDS-PAGE and LC-MS/MS, respectively. The results showed that we got the proteins of the salivary glands successfully.(3) Protein identification of the saliva and the salivary glandsBioinformatics method was used to analysis all the data acquired from the mass spetrometer. Protein identification from the MS/MS data was performed using the Mascot algorithm to correlate the data against the A. pisum protein database.We identified 141 proteins in the salivary gland and 31 in the saliva. 17 proteins are common, and they are the main saliva proteins. The results showed that both methods can get significant protein data and they can corroborate each other.(4) The analyse of saliva proteinComparing the saliva protein of the S.chinensis with three non-galling aphids, we finded that there was one common protein-oxidoreductase.It indicated that the oxidoreductase is the bascic function protein in saliva. It also indicated that the method we used is of good stability. The saliva of the S.chinensis has two specific binding proteins, which dominanted a high proportion. Thus we presumed that these specific binding proteins may be in relation to the gall formation. That should be the key point in the research of galling formation.(5) The analyse of salivary gland proteinThere are 6 proteins in common in the salivary gland secretome of S.chinensis and A.pisum.It indicated that the method we used is of great stability.Among the 141 proteins, there are 105 proteins with annotation and the proportion of binding protein is the highest, which was in keeping with the saliva. There are four kinds of specific proteins with a high proportion,which were presumed to be in keeping with the formation of galling. |
