Profiling And Peptide Mass Fingerprinting Of Proteins From Regenerating Tissues Of Girdled Trunk In Populus Tomentosa Carr.

Forest biologists take Populus as a model in the study of xylogenesis in hardwood trees, many studies on the fields of its cytology have been conducted. Recently, this model tree becomes more and more importance in studying molecular biology of xylogenesis. Populus tomentosa Carr. (section leuce) is a native species in China, playing a key role in wood production along the Yellow River and environment improvement in The Northern China. Therefore we have chosen Populus tomentosa Carr. as the material in our study.Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to obtain the protein profiles of the regenerating tissues after girdling the trunk of P. tomentosa trees. Matrix-Assisted Laser Desorption lonization Time-of-Flight(MALDI-TOF) Mass Spectrometry technique was employed to generate the peptide mass fingerprinting (PMF) of the differentially expressed proteins. The result will help to identify which genes regulate the activities of vascular cambium in trees, thus add new knowledge on wood formation. This would provide the basis for improving wood quality by genetic engineering. The main results are summarized as the following:We have set up the girdling and regeneration system for the trunk of P. tomentosa trees according to the technique developed for Eucommia Ulmoides. This system simulated in vivo the processes of cambium occurrence, cell differentiation and xylogenesis. We analyzed the protein changes of regenerating tissues at different developmental stages (i.e., 6*day, lO^day, 14*day, 18*day, 22ndday, 26*hday, SO^day after girdling) by 2D-PAGE technique with silver staining. We found 1370 differentially expressed proteins during this developmental process, showing remarkable dynamic changes of the proteins in the regeneration process.Many of these differentially expressed proteins found in this study belonged to low-rich proteins. In order to obtain enough protein in quantity from gel for MALDI-TOF MS analysis, we used Coomassie brilliant blue to stain the gel to isolate proteins changed in the regeneration process. 550 differentially expressed proteins were obtained, and 205 of which had the PMFs generated successfully.We performed database search for the 65 differentially expressed proteins from the 22th day's tissue (vs 18th day's). Most of proteins hit putative proteins, and only a few showed some similarity with known proteins in the database. In order to identify these putative proteins, further analysis such as protein sequencing and functional characterization needs to be done.