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Proteomics Analysis of Wing Disc from Silkworm, Bombyx Mori

Author: HanBin
Tutor: YiYongZhu
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: Bombyx mori Proteomics Wing disc Scaleless wings mutant Two-dimensional electrophoresis
CLC: Q966
Type: Master's thesis
Year: 2010
Downloads: 78
Quote: 2
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Abstract


There is a great variety of decorative pattern on the surface of the lepidopteran adults’wings. Patterns formed by multicolored scales is the main form of mimesis and protective color, it has a great physical importance to Lepidoptera. Wing disc is the organ of larve which will developed into the wings of adults; during pupation both the shape and the function of the wing disc will see definite changes. In this thesis, we used two-dimensional gel electrophoresis (2D) and biological mass spectrometry to research the protein pattern of silkworm wing disc during pupation. The result is very important to improve the understanding the development and the mechanism of control and regulation of wing disc. Further more, we compared the 2D profiles of scaleless wings mutant and a near isogenic line of scaleless (WT), and found out the differential expressed protein spots and did mass spectrometry identification. All the purposes were tried to clarify the mechanism of the mutant and offer references for the studies on the development of lepidopteran wing scales.1、The proteomics of silkworm developed so fast, and the 2D PAGE profiles of some organs had been established, like silk gland, midgut, fat body, colleterial gland and so on. But the 2D of insect wing disc has a few reports. We tried different lysis buffers to extract protein in the research and optimized the 2D condition. The result showed that higher protein extraction rate and resolution, prefect repetition were acquired in the present experiment.2、In order to research changes of the protein expression pertain of silkworm wing disc during pupation, we dissected the silkworm wing disc of the day before pupation (S4), the first day (P0) to the fourth day (P3), the seventh day (P6) and the day before eclosion, then we extracted the proteins and separated by two-dimensional electrophoresis. Chose P0 as control, compared others development phase with it, and we found out the changes. Compared wit P0, S4 has 39 newly expressed protein spots; P1 and P2 have 2 new spots each; P3 has 3 new protein spots; there are 32 and 48 newly expressed protein spots in P6 and P10 respective. Meanwhile, compared with the other six phases, P0 has totally 95 new protein spots expressed. Taking one with another, during the whole development, the newly expressed protein spots’molecular weight changed from high to low, and isoelectric point changed from acid to alkaline.3、To clarify the mechanism of the mutant, we compared the protein expression pertain of scaleless with wild type. Through software analysis, we found that the 2D image of both scaleless and WT (a near isogenic line of scaleless) in S4 are very similar; there is no specially or differentially expressed protein spot. The differential appeared from P0, in P0 there are 9 special spots especially expressed in the wing disc of WT with 15 proteins down-regulated and 4 proteins up-regulated more two times in scaleless. In the image of P1, 2 spots down-regulated and 1 proteins up-regulated in scaleless. In the third day after pupation, there are 3 spots down-regulated and 1 spot up-regulated. In P3, 3 spots of scaleless up-regulated more than two times. In the seventh day after pupation, there are 2 special spots especially expressed in the wing disc of WT with 3 spots up-regulated and 5 spots down-regulated in the image of scaleless. The day before eclosion, compared with WT there are 11 protein spots down-regulated and 6 up-regulated in scaleless more than three times.LC-ESI-MS/MS was used to identify the differentially expressed protein spots during the pupation of silkworm. Finally we identified 36 proteins, most of which belongs to curicle protein, and they account for 69.4% of all identified spots. Spot P0-23 is imaginal disk growth factor (IDGF), spot P0-25 is actin-depolymerizing factor 1(ADF1) and spot P0-26 is profiling. In the first day after pupation, IDGF up-regulated 2.6 times in WT wing disc, during the same day ADF1 and profilin respectively down-regulated by 2.3 and 2.2 times, and all of this three kinds of proteins effect the cellular motility and cytoplasmic extension, so we speculated that theirs coaction counteract the correct organization of tracheal system in the wing disc, and eventually caused the mutation of wing scale development.

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CLC: > Biological Sciences > Entomology > Insect biochemistry
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