| Pinus tabulaeformis Carr. is one of the primary species used in greening and afforestation throughout China. As the slow and significant development of its ovule, P. tabulaeformis is also a good material to the study of gymnosperm ovule development. The stage of female gametophyte free nuclear cellularization is one of the important stages during the ovule development. Regulated by functional genes, the cell wall of free nuclear is constructed in this period, which directly influence the quantity and quality of the seed. Protein is product of functional gene, which directly reflects the biological function of gene. Differential proteomic study on this process could directly explore the molecular mechanism of ovule development in P. tabulaeformis. In this dissertation,2D-DIGE combined with cytology, plant physiology and molecular biology methods were used to analysis the differentially expressed proteins in female gametophyte free nuclear cellularization of P. tabulaeformis. The major results were as follows:1. The changes of microstructure and ultrastructure in female gametophyte free nuclear cellularization were observed. The results showed that, the cell wall of female gametophyte is accumulated from scratch, and gradually formed and thickened. Cytoplasm, endoplasmic reticulums and golgi bodies were gradually increased during the cellularization process. Mitochondria were in rich content in the process. In the anaphase of cellularization, lip id droplets accumulated, indicating a strong protein and lipids synthesis happened in this stage. Hydrogen peroxide accumulation were observed in apoplast near the developing cell wall in this process. It speculated that hydrogen peroxide may affect the accumulation of cell wall.2. To separate the differential expressed proteins in female gametophyte cellularization, the 2D-DIGE protocols was optimized, the ovule proteins were extracted with TCA-acetone and rinsed with ice-cold acetone at least five times. The protein concentration of the sample solutions was adjusted to above 1.5 g·L-1, and the pH of the solutions was kept at 8.5 before labeling. This work established an available 2D-DIGE technique for proteomics research on female gametophyte free nuclear cellularization of P, tabulaeformis, and provided a technical reference for 2D-DIGE in tissues that are rich in secondary metabolites.3. Differentially expressed proteins during female gametophyte free nuclear cellularization was analysed with the optimized 2D-DIGE technology, and 81 differentially expressed protein spots were obtained. During the cellularization process,45 proteins were up-regulated,23 proteins were down-regulated,13 proteins reached the maximum expression in metaphase.47 proteins were successfully identified by MALDI-TOF-MS. Among them,7 proteins belonged to the cytoskeleton related protein,5 involved in resistance to oxidative stress,11 of them associated with secondary metabolites and lipid synthesis,5 involved in carbohydrate metabolism,9 involved in gene transcription, translation and protein degradation,2 belonged to cellular signal transduction.4. In the female gametophyte free nuclear cellularization process, Pre-mRNA-splicing factor CWC22, RNA helicase,26 S and 20 S proteasome were up-regulated expression, in contrast, histone H2Awas down-regulated. This indicates an increasing gene expression and protein synthesis function in the process. Serine hydroxymethyl transferase, glutamine synthetase, isoflavone reductase, citrate synthase, hydroxyacyl-ACP dehydration and other enzymes involved in secondary metabolites and lipid synthesis were up-regulated expression. Meanwhile, the expression level of enolase, Fructose-bisphosphate aldolase, triosephosphate isomerase and other enzymes involved in carbohydrate metabolism were also up-regulated. This results shows an active synthesis of storage substance in the anaphase of cellularization. Actin, a-tubulin, β-tubulin and other cytoskeleton associated proteins up-regulate the expression. The function of cytoskeleton maybe involved in cell wall material transportation or positioning. In this process, ferredoxin-NADP reductase, dihydrolipoy 1 dehydrogenase and catalase were up-regulated expression, which indicates the regulatory role of hydrogen peroxide in female gametophyte free nuclear cellularization of P. tabulaeformis.5. By determination the activity of antioxidant enzymes, the expression patterns of catalase and superoxide dismutase were validated. Meanwhile, the peroxidation level of protoplast membrane lipids was found to rise in free nuclear cellularization. The crucial role of catalase-2, which stabilized the intracellular reactive oxygen content during the cellularization process, was determined. The gene expression patterns of superoxide dismutase, catalase-2, catalase-3, ribulose 1,5-diphosphate oxygenase/decarboxylase were explored by reverse transcription PCR, which further verified the results of aforementioned proteomics study. |
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