Studies On EC Long-term Subculture And Proteomics During Somatic Embryogenesis In Litchi Chinensis Sonn

In this experiment, embryogenic callus (EC), transgenic resistant embryogenic callus(TREC) and embryogenic callus derived from transgenic protoplasts (TPEC) were used as the materials for the following studies in litchi : long-term subculture and synchronization regulation of somatic embryogenesis; induction of somatic embryogenesis based on the high-frequency regenerated technique of TPEC, which established by predecessor; further research of the differences of diverse cell lines and the expression of GUS in litchi by RAPD and GUS detection; two-dimensional gel electrophoresis analysis of proteins of cell line Q81.535 of litchi TPEC in the MS media adding ZT or without it during somatic embryogenesis in litchi; MS determination (MALDI-TOF-MS + MS/MS ) and functional analysis of somatic embryogenesis in litchi related proteins. The main results were as follows:1. The long-term subculture and the difference of different types cell line of litchi ECEmbryogenic callus (EC), transgenic resistant embryogenic callus(TREC) and embryogenic callus derived from transgenic protoplasts (TPEC) were subcultured and maintained by predecessor in litchi, then prolonged for 3 years. Besides, embryogenic callus still present primrose yellow, eugonic, granule petty and incompact, and maintained well capability of embryogenesis. In addition, part of cell lines of litchi TREC and TPEC were detected by GUS histochemical assays, it indicated that TREC cell lines Q811 and Q81.535 were steadily expressed GUS; TREC cell lines Q9223 were chimerically expressed GUS; TREC cell lines Q1016 were steadily unexpressed GUS. Among different TPEC cell lines , TPEC cell lines Q811 and Q81.535 were steadily expressed GUS; TPEC cell lines Q9223 were chimerically expressed GUS; TPEC cell lines Q1016 were steadily unexpressed GUS. The difference of diverse cell lines in appearance (shape, color and luster) and growth vigor were compared, then through RAPD analysis in part of cell lines of TREC and TPEC in litchi, it indicated that the difference was not obvious.2. Changes of the number of total proteins in somatic embryos (SE) during somatic embryogenesis in litchiLinear IPG strips (pH 4-7) was used for isoelectric focusing, which was carried out in the Ettan IPGphorâ…¢(GE Healthcare), and standard SE 600 Rube vertical electrophoresis system (GE Healthcare) was used for SDS-PAGE. And then the gels after silver staining were scanned with Imagine Scannerâ…¢(GE Healthcare) scanner, after that images acquired were analyzed by Image Master 2D Platinum 6.0 software (GE Healthcare), and the results showed that the pattern of total protein number in SE during somatic embryogenesis in litchi was: there was a obvious rise at first, reaching the highest point (for MS 20d), and then a slightly decline , again rapidly decreased, reaching the lowest point (for MS 30d), afterwards it went through a rapidly increasing process and steadily maintain the condition. At the medium term of somatic embryogenesis, that is, during somatic embryogenesis culture for ZT 20 d-ZT 30 d, the number of proteins changed the most dramatically, which took on rapidly reducing at first and then rapidly increasing.3. Changes of isoelectric point (pI) of total proteins in SE during somatic embryogenesis in litchiChanges of the proportion of proteins belonging to pI 4-5 were not server following the different culture time and condition, the proportion basically belong to 10.00%-20.00%, and showed a slowly downward trend, after a rapidly rise, and then maintained steady, after decline again, rapidly rise , at last speedy downward; the proportion of proteins of pI 5-6 decline slowly at first and then rapidly increase, where after reduced slowly; protein proportion of pI 6-7 slowly increased at beginning followed by a rapidly decline, reaching the highest point (for MS 20d), and then maintained steady, after slowly downward, finally increased, and the general trend was similar to "N" curve.4. Changes of molecular weight (MW) of total proteins in SE during somatic embryogenesis in litchiDuring somatic embryogenesis in litchi, protein spots of 30 KD-45 KD had a similar changing trend with 30 KD-45 KD, which took on rapidly rise at first, then decline , again increased and downward , rise finally; however, proteins within 20.1 KD-30 KD showed a trend that rapidly increased, then slightly dropped, afterward rose, maintained steady, the changes were not evident; small molecular weight proteins within 14.4 KD-20.1 KD showed that there was an increase at first followed by a drop and then an increase again, the peak was located at the time of MS 40d, then rapidly reduced; large molecular weight proteins within 66 KD-97 KD showed a trend that increased at first followed by a slightly decline, then basically maintained unchanging.5. Changes of the number of small molecular weight proteins (<= 15 KD) in SE during somatic embryogenesis in litchiDuring somatic embryogenesis in litchi, the number of small molecular weight proteins first increased and then significantly decreased, thereafter slightly rose, drastically downward finally. Whereas, the proportion of small molecular weight proteins had different variation patterns from the number of these proteins, which showed a slightly decline at the beginning and then a rapidly rise, after rapidly decline followed by speedily increased, then declined finally. However, at MS 40d somatic embryogenesis in litchi stage, regardless the number and the proportion of small molecular weight proteins in SE reached the peak (10, 2.54%), and proteins (<= 15 KD) accumulated the most at the time.6. Changes of the expression of the associated proteins during somatic embryogenesis in litchiDuring normal somatic embryogenesis in litchi with ZT medium, the expression of energy and carbon metabolism associated proteins, cell composition and the signal transduction associated proteins was first reduced and then tended to increase; the expression of the stress response and redox, lipid metabolism associated proteins showed a trend that progressively increase; the expression of protein metabolism and repair and amino acid metabolism associated proteins was increased at beginning then decreased; the expression of the nucleic acid metabolism associated proteins took on the trend that first decline then rise, and finally downward.During somatic embryogenesis in litchi without ZT medium cultured for 20 d, the expression of energy and carbon metabolism associated proteins, cell composition and the signal transduction associated proteins, the nucleic acid metabolism associated proteins, lipid metabolism associated proteins and protein metabolism and repair associated proteins was stronger than that under ZT medium cultured for 20 d; in the condition of ZT 20 d , the expression of the stress response and redox and amino acid metabolism associated proteins was stronger than that without ZT medium cultured for 20 d.7. Functional analysis of proteins related to somatic embryogenesis in litchiDuring somatic embryogenesis in litchi, mass spectrometry (combined MALDI-TOF-MS + MS/MS )succeeded in determining 19 proteins, added to Li Huan Ling(2009) 10 proteins which succeeded in determining, among these 29 proteins: except for 20.69% of the proteins with unknown function, 27.59% participated in stress response and redox, 24.14% of the proteins belonged to protein metabolism and restoration related, 13.79% of the proteins were involved in energy and carbohydrate metabolism, 3.45% of the proteins were associated with signal transduction, 3.45% of the proteins were of enzymes involved in nucleic acid metabolism, 3.45% of the proteins were involved in lipid metabolism, 3.45% of the protein were related to amino acid metabolism.