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Analysis of Antenna Protein of Silk Moth by Two -dimensional Electrophoresis

Author: WangGuoBao
Tutor: ShenWeiDe
School: Suzhou University
Course: Special Economic Animal Feeding
Keywords: Bombyx mori antenna two-dimensional electrophoresis MALDI-TOF/MS qPCR
CLC: Q966
Type: Master's thesis
Year: 2011
Downloads: 23
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Abstract


Antenna is the major communication organ among insects. Antenna have some male and female gender differences in external shape, smell and taste feeling mechanism, communication among insects and development. Currently, the molecular and physiological basis about these differences is not comprehensive. To explore the molecular basis of these differences, we used silk moth as a model insect, and the proteomics technologies were performed to research the protein expression profile characteristics of antenna of male and female silk moth by proteomic techniques, and obtained different and specific proteins between male and female antenna. And then using mass spectrometry techniques, we identified these proteins. On top of it, the characteristics of protein in the level of gene transcription characteristic was studied by quantitative real time PCR(qPCR) method. The results obtained are showned as follows:1 Research of protein expression profile of antenna protein of silk moth1.1 The establishment of patterns of antenna protein of silk moth by two dimensional polyacrylamide gel electrophoresisWe obtained expression profiles of total antenna protein and male and female antenna protein. The Image Master 6.0 software was used to analyze the two-dimensional gel electrophoresis profiles. Approximately 550 spots were detected in the antenna of silk moth, most of which were arranged from 14 to 70 kD with pI value(4~8). 419 and 489 spots were detected in the two-dimensional gel electrophoresis profile of female and male antenna at 4 minutes of color-display time, respectively, and the matched protein spots were 326. The matching rate was 71.81%.We obtained profiles which were clearer and have more protein spots by chananging color-display time. The Image Master 6.0 software was used to analyze the new two-dimensional gel electrophoresis profiles. 693 and 782 spots were detected in the two-dimensional gel electrophoresis profile of female and male antenna, respectively, and the matched protein spots were 513. The matching rate was69.56%. There were23 spots which have different gray value but distributed in both gel electrophoresis profile of female and male antenna. Meanwhile, there were 4 and 7 special spots which detected in gel electrophoresis profile of female and male respectively.1.2 The spectrum identification of different and specific protein in male and female antennaThe different and specific protein spots were identified by MALDI-TOF MS. Through database, we obtained nine proteins: imaginal disk growth factor, cuticular protein RR-1 motif 15, thiol peroxiredoxin, vacuolar ATPase B subunit, gasp precursor, mature 30K lipoprotein, abnormal wing disc-like protein, transketolase and chain A, solution structure of the Bombyx Mori pheromone-binding protein fragment. Imaginal disk growth factor, cuticular protein RR-1 motif 15, thiol peroxiredoxin, vacuolar ATPase B subunit, gasp precursor and mature 30K lipoprotein have higher expression levels in male antenna than in female antenna. Abnormal wing disc-like protein and transketolase have higher expression levels in female antenna than in male antenna. Chain A exist in all male and female antenna, but its position in the profile of male antenna was inconsistent with that in the profile of female antenna. These proteins are involved in different metabolic pathways respectively.2 Quantitative analysis of transcription levels of Bmidgf, Bmtpx and BmvatBThe transcription levels in each tissue of Bombyx mori of day 3 of 5th instar and in antenna of different periods of Bmidgf, Bmtpx and BmvatB were studied by quantitative real time PCR(qPCR) method. The detection results were normalized by using a Bombyx mori internal reference gene namely Actin3. The results indicated that Bmidgf, Bmtpx and BmvatB have different transcription levels in different tissues of Bombyx mori of day 3 of 5th instar. The transcription levels of Bmidgf and BmvatB have no obvious differences in antenna of different periods. Bmtpx have different transcription levels in antenna of different periods. It is thus concluded that the result was probably involved in the different function of antenna in different periods.We have analyzed the protein expression profiles of male and female antenna protein of silk moth. Through mass spectrometry we obtained ten kinds of protein which were expressed differentially and specificly between female and male antenna, including imaginal disk growth factor, abnormal wing disc-like protein and chain A etc. These proteins are involved in different metabolize mechanism, and probably related with the development of antenna of silk moth and the communication mechanism between male and female silk moth. The results not only enriched the proteomics research of Bombyx mori, but provided some basic informations for further study of morphological and functional differences between male and female antenna.

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CLC: > Biological Sciences > Entomology > Insect biochemistry
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