Studies On Proteomics During Friable Embryogenic Callus Development In Dimocarpus Longan Lour.

In this experiment,friable embryogenic callus(FEC) were used as the materials for the following studies in longan (Dimocarpus longan Lour. cv. Honghezi) :⑴two-dimensional gel electrophoresis analysis of proteins in friable embryogenic callus during normal development;⑵MS determination (MALDI-TOF-MS + MS/MS ) and functional analysis of friable embryogenic callus related to proteins.⑶to investigate the different growth condition of friable embryogenic callus growing on the MS media with different concentrations of polyamines (spermine,spermidine,putrescine),to understand well the growth and aging mechanism of longan FEC,which supplied scientific basis for cultivation, long-term subculture and keeping embryonic . The main results were as follows:1 Changes of the number of total proteins in FEC during the normal development in longanDuring the normal development in longan, the number in 2-D map of total proteins ranged from 400 to 800 , and the pattern of total protein number in FEC during friable embryogenic callus development in Longan was: there was a slight rise at first, and then a obvious decline, reaching the lowest point (for 30d)- associated huge changes in number, afterwards rapidly ascending,again, it went through a slight rise process and the number of proteins go up to the highest value (for 50d).2 Changes of isoelectric point (pI) of total proteins in FEC during the normal development in longanIsoelectric point of all proteins was distributed between 4 and 7. Changes of the number of proteins comprising pI values ranging from 4 to 5 and 5 to 6 were similar,showed a obvious rise,after a decline,then increased again, the overall variation trend was similar to "N" curve, nevertheless, the protein number of pI 5-6 reached the greatest value before pI 4-5, moreover,the number of proteins was always more than which ranged from 4 to 5; conversely,the number of proteins of pI 6-7 decline slowly at first and then rapidly increase, reached its highest point for 40 d, finally slowly downward.3 Changes of molecular weight (MW) of total proteins in FEC during the normal development in longanMolecular weight of proteins of FEC during the normal development in Longan was within 14KD-97KD. However, the variation tendency was different, protein spots of 30 KD-45 KD showed a antipodal changing trend with which form 45 KD-66 KD, which took on rapidly rise at first, then maintained steady, otherwise, proteins varied from 45 KD to 66 KD kept stable after rapidly descent. proteins ranged form 14.4KD-20.1KD and form 20.1 KD-30 KD were slightly increased at beginning followed by slowly dropped ,then increased again. The changes of proteins within 66KD-97KD were not evident,showing a slightly decrease for 50d.4 Changes of the number of small molecular weight proteins (<= 15 KD) in FEC during the normal development in longanDuring FEC development in longan the total number of small molecular weight proteins varied from 5 to 20. The tendency of the number of small molecular weight proteins first decreased slightly and then significantly increased,till smoothed finally. Moreover, the proportion of small molecular weight proteins showed a similar variation patterns with the number of these proteins, which showed a slightly decline at the beginning and then a obvious rise, thereafter slightly declined again. And the proportion of small molecular weight proteins in FEC reached the peak (2.68%) at 40d.5 Functional analyses of proteins related to FEC development in longanPercent twenty-eight point seventy-nine of the 66 identified proteins (added to He(2009)&Lai(2010)18 protein spots) related to FEC development in longan were proteins with unknown identity or function. 30.30 % of the proteins participated in stress response and anti-oxidation;10.61 % of the proteins were involved in energy and carbohydrate metabolism;10.61 % of the proteins belonged to protein synthesis related; 6.06 % of the proteins were of structural proteins involved in cytoskeleton remodeling; 4.55% of the proteins were regulatory proteins; additionally 1.52 % of the proteins were associated with signal transduction and PCD,1.52 % of the proteins were related to amino acid metabolism; 1.52 % of the proteins were lipid metabolism-related protein;3.03 % of the proteins were of enzymes involved in nucleic acid metabolism;1.52 % of the proteins attached themselves to vitamin metabolism.6 Changes of the expression of function-related proteins in FEC during the normal development in longanIn the early stage(10-20d),the expression of redox proteins associated with FEC in longan (including ascorbate peroxidase R147, R158 and HC21, phenylcoumaran benzylic ether reductase homolog TH6, glutathione peroxidase and peroxidase L26) was high,while the expression of peroxidase 4 was in late stage (40-50d).Protein synthesis–related proteins showed a expression peak for 20d,besides protein isoform 1 for 30d. Different energy-associated proteins expressed more abundantly during whole period. S-adenosylmethionine synthetase and peroxidase associated to polyamines were rich in FEC at late stage. Polyamines could remove reactive oxygen species like proxidase, moreover improve the activity of antioxidase.7 Different growth condition of FEC growing on the MS media with different concentrations of polyamines(PAs)On the basis of identification of the enzymes-related polyamines by proteomics technology in FEC, further study was done,which was on the effects of exogenous PAs (spm,spd,put)on the growth of FEC in longan.The results were as follows,the characteristic of FEC showed complete browning, and however, some of them were proliferating and kept living. The FEC cultivated on different media with spm (1mg/L, 7mg/L, 9mg/L) or spd(3mg/L,7mg/L),or put(9mg/L), compared to the CKr medium, whose wight continued increase . The cells viability of FEC cultured on the media with spm(3mg/L, 5mg/L) or spd(9mg/L)was enhanced except put.