Establishment Of The Two-dimensional Electrophoresis System Of Somatic Embryo In Lycium Barbarum And Preliminary Analysis Of Its Protein Expression Profile

Plant somatic embryogenesis is one of the fundamental development pathways during plant tissue and cell culture in vitro. Here, Lycium barbarum L is used as the experimental material in the research. At first, the rapid and efficient induction system of somatic embryo was established by use of different explants and different auxin concentrions. Based on this, we adopted the relevant techniques of proteomics to analyze the specific genes which express during the early stage of Lycium barbarum L somatic embryogenesis at the level of translation. We respectively extracted the proteins of embryonic callus (EC) and somatic embryos (SE), compared the advantages and disadvantages of the three kinds of extraction methods—direct extraction, precipitation and TCA-acetone precipitation, discussed impact of the" different Isoelectric focusing (IEF) parameters on the effect of electrophoresis. Quantity one software was used to quantify and examine the gel map, which facilitated to find the differential protein spots between EC and SE .The differential protein spots were identified by MALDI-TOF-MS and the results were searched in the database. The results were as bellow:1. The hypocotyl of Lycium barbarum L sterile seedlings could be induced embryonic calli with the best quality when cultured on MS induction culture medium added 2, 4-D 0.4mg/L, meawhile, when transferred to auxin-free MS culture medium it can produce numerous somatic embryos.2. TCA-acetone precipitation was regared as the most effective methods to extract proteins due to the higher protein content and better electrophoresis effect which payed a good foundation for the establishment of the two-dimensional electrophoresis system .3. Differential proteins between non-embryonic callus and somatic embryos were detected, and analysis of the differential strips had been done.4. 244 and 256 protein spots were respectively found in EC and SE. Differential protein spots were examined by 2-DE software. Among them two obvious and reproducible protein spots were identified by MALDI-TOF-MS, and partial amino acid sequences were obtained by searching database. 5. Bioinformatics technique, including MASCOT, NCBI database, expasy protparam analysis and expasy ProtScale, were used for searching the potential sequence of the proteins above and their corresponding genes.Taken together, an efficient induction system of somatic embryo on Lycium barbarum L was established. And the proteins related on somatic embryo induction were separated and analyzed via various proteomics technologies, and partial amino acid sequences of peptides were obtained. The results above may explain the somatic embryogenesis on the level of proteomics and pay a good foundation for the molecular study related on somatic embryogenesis. Our research may throws a light on the molecular mechanism of somatic embryogenesis and offer clues for gene cloning by predicting gene. sequence according to the amino acid sequences.