Localization Of The Type Ⅱ Metacaspase Protein(TaeMCAⅡ) And Proteomic Analysis In Sieve Elements Development Of Triticum Aestivum L.

Programmed cell death controlled by the gene which is the death of cells in an actively and orderly manner. Metacaspases, the caspases-like protease, may play an important role in plant PCD. Sieve elements(SEs) development was proved to be a special PCD process in the abdominal vascular tissue at wheat caryopsis. The mature SEs not only keeps the cell activity, but also exerts the function of nutrient transport. This study focuses on PCD features, as well as the type II metacaspase protein(Tae MCAII) gene expression and its dynamic distribution during the developmental process of wheat SEs. In order to further explore the function protein during the SEs development of the abdominal vascular tissue at wheat, so as to provide the theoretical foundation for the subsequent research, we adopt protein two-dimensional electrophoresis and mass spectrometry technology to detect the correlation protein. The research mainly results are as follows:1. The trastructure displayed the cell number with the SEs development, and the unltrastructure revealed the dynamic changes of the wheat SEs development at the subcellular level, such as enucleantion, inclusions missing, cell wall thickening and cytosol density decrease the subcellular level. The SEs development process similar to PCD by showing PCD-like cell organelles degradation. But in the late development, the cell kept some organelles helping the SEs remain active.2. The TUNEL found that there was no positive labeling(indicated by green fluorescence) at 2 DAF, 5 DAF, 6 DAF and 7 DAF. The green fluorescence was only observed at 3 DAF and 4 DAF indicating that the nuclear degradation happened at these periods.3. The q-PCR and situ hybridization revealed that the transcriptional level of Tae MCAⅡ m RNA in the SEs development observed the following changes: first displayed an increasing trend and reached a much higher level at 3 DAF, and then showed the decreasing trend except a sharp increase at 7 DAF. This indicated that Tae MCAII still functioned at the late stage of SEs development.4. Immunohistochemical observation further revealed the dynamic changes of Tae MCA II from a protein level. It was found that Tae MCAII was mainly detected at 3 DAF, 4 DAF and 7 DAF indicating the key roles played by Tae MCAII in these stages. Furthermore, Immuno-electron microscopy revealed that Tae MCAII was first localized in nucleus, and then cell cytoplasm was also observed, and finally, it was mainly detected on cell membrane and cell wall of late-stage SEs.5. The analysis of the dynamic changes of expressed proteins in developmental SEs at 3 DAF, 5 DAF and 7 DAF by proteomic, 2-DE statistical analysis revealed 126 differentially expressed protein spots with at least a two fold change(P<0.05). By further manual validation and quantification, 86 spots were selected to be excised from the 2-DE gels and 80 spots were identified by mass spectrometry, which 6 of them might be the same one and also 2 unknown proteins.Above all, SEs development in wheat appeared the typical characteristics of PCD, such as the nucleus degradation, inclusions missing and so on. But mature SEs remains a few organelles and the cytoplasm indicating that the cell still keep activity so as to functioning as a photosynthesis product transportation access. TUNEL detection showed that the nucleus DNA rupture occurred in 3 DAF and 4 DAF. Tae MCAII distribution characteristics in the process of SEs development are consistent with the process of SEs PCD. These data indicated that Tae MCAII might play an important role in the PCD-like process of SEs development. In order to study the proteomic analysis of wheat caryopsis SEs functional proteins during the development, two-dimensional electrophoresis technology was used to separate the differences proteins of the three key period, then followed by mass spectrum identify these proteins which the expression level with more than two-fold changes(P < 0.05) were choosed. By above approach, a total of 80 differentially expressed proteins were obtained, and the protein correlation analysis is still under analysis.