Proteomics During Somatic Embryogenesis In Longan (Dimocarpus Longan Lour.)

In this experiment, the LC2 embryonic cell line of the cultivar "Honghezi" of longan (Dimocarpus longan Lour.) was used as materials for obtianing the synchronized embryogenic cultures of somatic embryogenesis at different developmental stages and further researches were as follows: â‘  analysing the specific proteins expressed during longan somatic embryogenesis by 2-DE; â‘¡ separating the low-molecular-weight (LMW) peptide expressed at the late-stage of somatic embryogenesis; â‘¢ identifying the peptide by Peptide Mass Fingerprint (PMF) and MS/MS, and cloning the related gene by RT-PCR; â‘£ analysing the factors affecting the expression of the LMW peptides during longan somatic embryogenesis. The results were as follows:1. 2-DE analysis of the specific proteins expressed during longan somatic embryogenesis. The 2-DE analysis of the total proteins of the embryonic callus, globular embryo, cotyledonary embryo, early-stage mature embryo, mid-stage mature embryo and late-stage mature embryo of longan showed that: â‘  the embryonic callus of longan expressed the most varieties of acidic proteins (the value of pI was between 4 and 5), which eliminated gradually in the process of development of somatic embryos, on the opposite, slightly acidic proteins (the value of pI was between 5 and 6) increased gradually; â‘¡ during late developmental stages, the high-molecular-weight (HMW) proteins eliminated gradually, and the low-molecular-weight proteins simultaneously appeared and accumulated gradually; â‘¢although there were specific spots at each stage, the general diversificational trend was obviously decreased gradually; â‘£ there were some differential spots standing for specific proteins.especially at the late-stage somatic embryogenesis, a series of high-molecular-weight proteins disappeared, and a series of low-molecular-weight proteins appeared, which included some highly expressed proteins that were probably closely associated with somatic embryogenesis.2. Separation and identification of the specific LMW peptide during longan somatic embryogenesis. The SDS-PAGE analysis of the total proteins of the embryogenic callus, globular embryo, heart-shaped embryo, torpedo embryo, cotyledonary embryo, mature embryo of longan showed that the molecular weight of the LMW peptide discovered only in mature embryos was lower than 14.4 kD, and at present there was no report about a specific protein with so low molecular weight expressed during plant somatic embryogenesis except for longan.The classical method of SDS-PAGE was not suitable for separating the peptide whose molecular weight was lower than 15 kD, and the improved method of Tricine-SDS-PAGE was used to separate the low molecular weight peptide and the ideal result was obtained. The peptide was well separated in the gel, the molecular weight was approximately 7.4 kD. The gel which included the peptide was cut off for Pepetide Mass Fingerprint (MALDI-TOF) identification, but there was not enough information in the result to make sure what the peptide was, and it was further identified by MS/MS (Q-TOF) Analysis, in the result, the sequence of a 8-amino acid residue-segment (AISTEKSK) in the peptide was the same as the segment of ATPase 8 subunit in Roeboides...