| Pollen tube, a carrier of sperm nuclei during the process of sexual plant reproduction in flowering plants, is a highly polarized,rapidly tip-growing plant cell, and thus is an ideal system for studying the molecular mechanism involved in the regulation of cell growth. In this paper, we used mature pollen grains(MP), hydrated pollen grains(HP) and pollen tubes(PT) of Populus simonii×P.nigra as materials. By cytological observations, Solexa sequencing, two-dimensional electrophoresis, liquid chromatography tandem mass spectrometry based on the non-labeled quantitative (Label-free LC MS /MS) techniques, we investigated pollen germination dynamics, germination rate and suitable in vitro germination conditions. The differentially expressed genes and proteins with their functional categories during pollen germination (from.MP to HP, referred to as PG) and pollen tube growth (from HP to PT, called PTG) were also studied. Results are summarized as follows:1. Mature pollen grain of P. simonii×P.nigra is 2-celled pollen. Pollen germination Observation:The germination rate gradually increases at 1,2,4,6,7 hours and up to more than 70% at 7 hours. The average length of pollen tube is 200～300μm2. The appropriate conditions for pollen in vitro genmination:Room temperature about 21℃, PH6.3, sucrose 15%, boric acid (H3BO3) 20 mg/L and calcium chloride (CaCl2) 40 mg/L3. Solexa sequencing evaluation:The highest number of Clean tags in three sample is 4.5M and the lowest one is 2.5M. All Tag Mapping to Gene accounted for about 50%. Unambiguous Tag-mapped Genes reached 10000 or so. New transcripts reached 30000～40000. The overall sequence data is normal4. There was a decline in the total number of genes transcripted from MP to HP and an increase from HP to PT. The proportion of specifically expressed genes decreased significantly from MP to HP and increased significantly from HP to PT. Down-regulated genes in differentially expressed genes during PG were dominant and up-regulated genes in differentially expressed genes during PTG were dominant5. Enriched analysis of Molecular function in differentially expressed genes shows:The transcripts involved in categories of catalytic activity, binding, transporter activity, emzyme regulator activity were overrepresented during PG and PTG6. Enriched analysis of Biological process in differentially expressed genes shows: Biological process related to PG mainly included transpoter, localization and establishmen of localization. Biological process related to PTG mainly included spingolipid metabolic process, membrane lipid metabolic process, alcohol metabolic process7. Pathway enriched analysis shows:There were 49 significantly enriched pathways during PG, such as calcium signaling pathway, phosphatidylinositol signaling, MAPK signaling, Inositol phosphate metabolism and so on. There were 24 significantly enriched pathways during PTG such as Sphingolipid metabolism, Galactose metabolism, Glycolysis /Gluconeogenesis, Ubiquitin mediated proteolysis and so on8. We obtained 612 differentially expressed proteins by Label-free LC MS/MS.301 differentially expressed proteins were remained after removing the redundant proteins, of which there were 5 MP-specific expressed proteins,155 HP-specific expressed proteins and 64 PT-specific expressed proteins. Specifically expressed proteins were significantly increased from MP to HP and decreased from HP to PT9.301 proteins were grouped 12 functional categories. Proteins related to carbohydrate and energy metabolism, protein metabolism accounted for about 50%. Proteins involved in defense or stress, cell wall construction and metabolism, transport, cytoskeleton dynamics accounted for approximately 30%10. Integration analysis of gene expression data and proteomic data shows that the correlation between the two groups of data was very low and even showed a reverse relation... |
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