| Silkworm is the typically mode insect in Lepidotera, which's hemolymph is charged with many important physiological function and important part in body fluid. Transported in tissue by hemolymph, Nutriment and hormone keep metabolize and development in body. Hemolymph is temporary metabolize place in silkworm, we could find out the mechanism of physiology and pathology efficiently while its protein changed. Silkworm Physiological structure obviously changed in moth under the control of some important hemolymph proteins. Because of moth do not taking food, the energy of its movement and mating is supported by storage protein and transport protein. Some low weight hemolymph proteins play many important roles in many special biologic behavior in moth. For hemolymph proteins expression distinctly different in stage, it is important for gene expression and development control in silkworm. Hemolymph proteomics study just on the start. By using two-dimensional polyacrylamide gel electrophesis and PMF, we identified some important proteins related with moth and compared some different protein in moth. We studied the function of ApolipophorinⅢprotein which expresses highly in moth primarily. In this thesis, the works which we did are showed as follows:1. Identification of hemolymph proteins By using two-dimensional polyacrylamide gel electrophesis and MALDI-TOF-MS in moth: By using two-dimensional polyacrylamide gel electrophesis, we established 2D map with hemolymph in moth. These spots mostly distributing in 15KD～90KD with pH 3.5～9.5. By PMF, we identified 38 spots of the better 50 spots in 2D map, which comprised five Chymotrypsin inhibitor: CI-8A,CI-2,antichymotrypsin precursor,antitrypsin precursor,inhibitor of apoptosis 1, three enzyme with metabolize: beta-N-acetylglucosaminidase isoform A,aminoacylase,hydroxypyruvate isomerase, two enzyme with immunity: Phenoloxidase subunit 1,hemolin, two Molecular chaperone: peptidylprolyl isomerase B,heat shock protein hsp20.1, protein with biologic behavior: diapause bioclock protein,dehydroecdysone3-reductase, one storage protein: Vg, three low molecular weight lipoprotein: ApolipophorinD,ApolipophorinⅢ,30k protein. dehydroecdysone3-reductase and ApolipophorinD are protein without report in silkworm. It is implied that hemolymph protein is concerned with complex biologic process in moth. 2. comparative analysis with different protein in moth: By using Image Master2D Platinum3. software to hemolymph 2D map with the 6th day in pupa stage and the first day in moth stage, we found that 97 spots were matched in the two 2D map and match rate was 54.3417％, and it is also showed that there are many different spots in the two 2D map. There are 6 observably different areas in the two 2D map. We found that they are CI-8, Vg, 30K protein, ApolipophorinD, diapause bioclock protein and ApolipophorinⅢ. These different protein expression are high in pupa or in moth, which is implied that the important biological responses to abnormality in moth.4. ApolipophorinⅢgene was cloned and its function analysis primarily: By MALDI-TOF-MS identified, we found that there are 186 amino acid in the protein sequence, translated 623bp length cDNA sequence, which's ORF length is 560bp, and the genome sequence with 1076 bp length has 4 exons and 3 introns by blast genome date. Successfully cloned the gene, we proved the veracity of MALDI-TOF-MS identification. The multi-alignment analysis show that the homology similarity is high in Lepidotera and low in orthoptera and hymenoptera, and in Lepidotera, the homology is similar in Sphinx except in Mandarina, so in evolution process, ApolipophorinⅢgene is conservative in Lepidotera.The expression of ApolipophorinⅢsuddenly increased before moth identified by SDS-PAGE electrophesis. It is show that ApolipophorinⅢis concerned with lipid transport in moth and playing the extremely vital role. From the mierorarry result analysis, in front of the emergence the ApolipophorinⅢgene expression quantity male is higher than female, the most possibility is male moth's flying ability must surpass the female moth.The RT-PCR examination indicated that, the ApolipophorinⅢgene expresses in many tissues, it may involves some other important unknown functions. Using the bombyx mori microrarry data, discovered in pupation period, the ApolipophorinⅢgene expression quantity has the obvious rise, by the hemolymph SDS-PAGE electrophoresis result, ApolipophorinⅢprotein does not synthesize and secrete in hemolymph in the period of pupation, extrapolated the ApolipophorinⅢprotein has synthesizes possibly in the epidermis, the fat body and the ovary and so on which are the mRNA level expression quantity high tissues. We concerned that it is related with the progress of apoptosis in pupa and development with Ovary.
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