Dissertation > Biological Sciences > Botany > Plant cytology

Cellular and Proteomic Analyses of Endocytosis Mechanism Involved in Pollen-tube Growth in Nicotiana Tabacum

Author ZhaoPengFei
Tutor WangWei
School Henan Agricultural University
Course Botany
Keywords Tobacco Pollen germination Proteome Two-dimensional electrophoresis MS Transmission electron microscopy
CLC Q942
Type Master's thesis
Year 2011
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ABSTRACT:Pollen germination and pollen tube development play a crucial role in sexual reproduction of flowering plants as pollen tubes deliver sperms into the ovary, a necessary process for fertilization and seed set. So, In many years its as the model of studying endocytosis ,cell division, differentiation and cell identification and other important life process. At present, The research of signal transduction in Pollen tube polarity growth has made great progress, but The proteome changing and identification of proteins involving in pollen germination growth were studied little.We have used 2-DE and LC-ESI-MS/MS to identify and analyze differentially expressed proteins during pollen germination in maize. The main results are represented below.1. We has established a 2-DE system which be suitable for maize pollen and compatible with mass spectrometry technology. In this study, for 2-DE, protein (400μg in 250μl rehydration buffer) was loaded into 11 cm linear pH 4-7strip, via passive rehydration overnight, the total value 55000vhr. Afterwards, SDS-PAGE was run in 12% polyacrylamide gel. Gels were stained with CBB R250. The 2-DE map is clear and the proteins which uniformly spread on the gel were round or oval shape, regularly, clear and good repeatability,can better satisfy follow-up mass spectrometry (ms) identification.2. Proteomic mapping of pollen germinated in vitro for different time intervals in the presence of endocytosis and/or cytoskeleton inhibitors. Protein identification by use of immunoblotting and MS/MS analysis to evalute the differentially expressed proteins. Among about 358 proteins separated in 2D gels, the abundances of 28 protein spots changed visibly (up- or down- regulation) between treatments and contrast experiments. These proteins represent 21 distinct proteins, of which 13 up-regulated, mainly involving in tube wall modification, actin cytoskeleton organization, energy metabolism, signaling, protein folding and degradation, the other 8 proteins were down-regulated, including pollen allergens and defense-related proteins. This study would contribute to the understanding of the changes in protein expression associated with pollen tube development.In conclusion, we have used 2-DE and LC-ESI-MS/MS to compare the change between germinated pollen gains and mature pollen gains, identified differentially expressed proteins during pollen germination in maize, and .This study presents useful molecular information at protein level to further understand the mechanisms underlying pollen germination and tube growth.

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