Proteome Changes Induced By Cold Acclimation In Callus Of Saussurea. Laniceps Hand.-Mazz.

The callus induced from the seed of Saussurea laniceps hand.-Mazz.which is distributed in the Tibet Plateau,4300-5280meters above sea levels, was used as the object of present study. Combined with bioinformatics research methods,2D-PAGE and mass spectrometry techniques were used to study the change of protein abundance when the callus was acclimated by low temperature, and to analyze the function of low-temperature-responsed proteins of the Saussurea.laniceps hand.-Mazz. This study provides the experimental basis to reveal the resistance mechanisms of Saussurea laniceps which is an endemic alpine plant grown in the alpine zone.The rich polyphenol, amylase and secondary substance accumulated in Saussurea laniceps because of the unique alpine habit can seriously interfere with the results of2D-PAGE. In this research, three protein preparation methods (TCA/Acetone extraction, Urea/thiourea extraction, phenol extraction) on the aspects of protein yield and protein purity of electrophoresis pattern were compared. The results indicate that the phenol extraction method is the most suitable for the preparing of callus proteins of Saussurea involucrate. Although the protein purity extracted by phenol method was less than TCA/Acetone-and Urea/thiourea-method, the purity of protein was29%. SDS-PAGE analysis showed that the protein bands extracted by phenol method were more and clear than the other two methods.2D-PAGE analysis showed that more than60point of protein extracted by phenol extraction was larger than the other method. The2D maps of phenol extraction had higher resolution and reproducibility than the other two methods. The most suitable protein quantity for17cm strip to2D-PAGE was500μg. Above works laid solid foundation for the alpine plant proteomics.The callus were induced at0℃for0h(control group),12h,24h,48h,96h,192h, respectively. Forty-one differentially expressed proteins were found and identified by the mass spectrum through using the PDQuest software to analysis the2D maps. Only8positive results were obtained because the Saussurea involucrate have not been sequenced and the incompleteness of database. Five positive results were up-regulated proteins (i.e., NADP-dependent isocitrate dehydrogenase, resistance protein RPP8-like protein, Adenosylhomocysteinase, Hypothetical protein [Oryza sativa (japonica cultivar-group)], Tubulin alpha chain). The rest3were down-regulated proteins (i.e., DNA-binding bromodomain-containing protein, glucose-6-phosphate isomerase, and Beta-tubulin). These differentially expressed proteins were found to relate to low temperature resistance based on the bioinformatics analysis. Finally, these41points were divided into10categories (i.e., metabolism(17.07%), protein synthesis(14.63%), stress(9.76%), transport(9.76%), cell structure(9.76%), energy(7.32%), photosynthesis(7.32%), protein degradation(2.44%), transcription(2.44%) and unknown(19.51%) based on their participation in the biochemical processes by using the UniProt database. It indicates that protein synthesis and metabolism play important roles in cold resistance of Saussurea.laniceps hand.-Mazz. callus.