| In this experiment, five early-different-stage embryogenic culutures obtained after synchronization regulation from the embryogenic callus (EC) were used as the materials for the following studies in longan (Dimocarpus longan Lour. cv. Honghezi):①establishment of the high-resolution two-dimensional electrophoresis technology system for analysis of the proteins from early-different-stage embryogenic cultures in longan;②separation and preparation of proteins from 5 early-different-stage embryogenetic culutures by two-dimensional electrophoresis, and the analysis of protein differential expression using the soft of ImageMaster 2D Platinum 6.0( GE Healthcare;③identification of parts of differential expression proteins from 5 early-different-stage embryogenetic culutures by mass spectrometry and analyses of their functions;④cloning of parts of early embryogenic cultures related protein genes with homologous cloning or degenerate primers according to the protein identified results, and detection of transcriptional expressions of these genes during somatic embryogenesis of longan by real-time fluorescence quantified PCR (qRT-PCR). The main results were summarized as follows:1. Establishment of high-resolution two-dimensional electrophoresis technology for the analysis of the proteins from early embryogenic cultures in longanThe synchronization of embryogenic cultures was carried out by regulating the concentration of 2, 4-D on the medium and microscopic observation as reported previously. Five early-different-stage embryogenetic culutures of longan were obtained including the friable-embryogenic callus (FEC), embryogenic callus II (ECII), incomplete compact pro-embryogenic cultures (ICpEC), compact pro-embryogenic cultures globular embryos (CpECGE) and globular embryos (GE). Immobilized pH gradients (IPG) two-dimensional electrophoresis was developed to investigate the separation of proteins from early embryogenic cultures of longan. The results indicated that the isoelectric points (pI) of proteins mainly focused on the pH value between 3.5 and 7.0. The better result was obtained under the condition of the pH value between 4 and 7 using same length IPG strips. The resolution was improved by the IPG two-dimensional electrophoresis. The numbers of protein spots separated using IPG two-dimensional electrophoresis with 13 cm strips were two and a half times of that using traditional two-dimensional electrophoresis, and many low abundance proteins could be detected. The resolution of proteins could be further improved with increasing of strip lengths. The numbers of protein spots detected in 18 cm gels strips was about two times of that in 13 cm gels strips. But a perfect 2-D map could be obtained by adjusting of isoelectric focusing parameters and with high quality gels of SDS-PAGE.2. 2-DE analysis of embryogenic cultures at early different stages in longan①The variation of soluble protein amounts of embryogenic cultures at early different stages during longan somatic embryogenesis. The soluble protein contents of embryogenic cultures at early different stages during longan somatic embryogenesis were detected by method of Coomassie brilliant blue G-250. The results showed that the soluble protein contents increased with the initiation of longan somatic embryogenesis. They were low in FEC stage and reached highest in ECII stage, then decreased slightly and remained at a higher level than that of FEC at subsequential stages. The soluble protein contents at FEC stage were extremely significant (P<0.01) lower than that of other stages, but there was little differences at other stages. These results suggested that the variation of soluble protein contents of different embryogenic cultures at early stages during longan somatic embryogenesis be related to development of somatic embryos.②Obtaining of 2-DE gels of the total proteins of five early-different-stage embryogenic cultures in longan and changes of the number of their total proteins. The average numbers of total protein spots decreased sharply at first and then increased greatly, subsequently decreased abruptly again. The spots of total protein were maximum at CpECGE stage with 1798 spots, and it reduced to a minimum with 1203 spots at ECII stage. Total protein numbers at other stages were 1461 (FEC), 1582 (ICpEC) and 1499 (GE) spots. It was concluded that the sharp fluctuation of total protein numbers of different embryogenic cultures may be related to specific expression of genes at early stage during somatic embyogenesis in longan.③Changes of isoelectric point (pI) and molecular weight (MW) of total proteins in different embryogenic cultures at early stage during longan somatic embryogenesis. The results showed that pI of most proteins ranged from 5 to 7. The average numbers of these proteins was more at last three stages than that at the first two stages. And the numbers reached the highest at CpECGE stage under any range of pH values. ECII stage had lowest numbers of proteins than other stages expect under the pI value with 6 to 7. Average number of proteins at ECII stage was lowest than that of other stages, but it had more proteins than FEC stage in the range of MW from 20.1 kD and 30 kD. The numbers of proteins with other MW at this stage were lower than that of other stages. Number of proteins with different MW at CpECG stage was more than that of other stages, except that proteins with MW larger than 66 kD. This implicated that ECII and CpECGE stages were critical for morphogenesis. Number of proteins with different MW decreased at GE stage, except that proteins with MW larger than 66 kD, which increased slightly at this stage. These may be related to shut down the expression of some genes involved in somatic embryogenesis, which was only obligatory at early stages.④Analysis of differentially expressed proteins in different embryogenic cultures at early stage during longan somatic embryogenesis. 148 differentially expressed proteins were selected for followed analysis. The results showed that 10 proteins (spots of 009, 044, 047, 060, 075, 090, 093, 094, 138 and 148) were specific expressed at different stages. Spot of 009 only expressed at CpECGE stage and GE stage. Spot of 044 was absent at GE stage, spot of 047 was absent at ICpEC stage, spot of 060 only expressed at FEC stage, ICpEC stage and CpECGE stage. Spot of 075 only expressed at ECII but spots of 090, 093 and 138 were absent at this stage. Spot of 094 only expressed at ICpEC stage and CpECGE stage. Spot of 148 expressed at FEC stage, CpECGE stage and GE stage. Most of the proteins were differentially expressed in rules, spots of 002, 010, 024, 043 and 078 were down regulated with early development of somatic embryo. Spots of 008, 028, 030, 35, 36 and 120 were opposite. Expression level of spots of 032, 033, 038, 039, 050, 055, 071, 089, 091, 119 and 121 were higher at FEC stage. They decreased at EC II stage, then increased with somatic embryo growth and reached the highest at GE stage. These suggested that the development of somatic embryo during the early stage be modulated by large sets of proteins.3. Identification of some differential expression proteins by mass spectrometry and analysis of their functions118 proteins were subjected to mass spectrometry (MALDI-TOF/TOF) analysis. 45 (37%) of them were identified, and they were classified to eight functional classes, which including 10 proteins involved in energy and glucose metabolism (22%), 3 proteins involved in signal transduction and programmed cell death (7%), 5 proteins involved in regulation (11%), 12 proteins involved in stress response and defense (27%), 2 proteins involved in cell structural (4%),3 proteins involved in protein synthesis and processing (7%), 1 proteins involved in amino acid metabolism (2%), and 9 unknown proteins (20%).Among of the identified proteins, 4 proteins were identified as enolases. Spots of 067 and 068 were identified as alnus enlases and spots of 069 and 070 were identified as soybean enlase. But they were located on the different sites in the 2D gels. Spots of 067, 068 and 069 were almost the same in molecular weight but different with pI. Spot of 70 was different from others. These proteins may be the different forms of the same protein which had different post translated modifications. Spots of 133 and 139 were identified as triosephosphate isomerase. These results suggested basal metabolism was activity at early stages of longan somatic embryogenesis, which was the basis of embryo development, but their expression levels were different which implicated that they may have other functions. Spots of 41, 66 and 100 were subunits of ATP synthase, Spots of 41 and 66 loclalized in mitochondria, and spot of 100 was subunit of vacuolar ATP synthase. These suggested that energy metabolism was activity during somatic embryogenesis. Spots of 111, 112, 113, 114, 116 and 117 were identified as tobacco peroxidase, spot of 115 was fiberflax peroxidase. Some of them had the same MW and some of them had the same pI. They may be different post translated modification products of peroxidase. Most of the identified proteins were related to oxidative stress response, which indicated oxidative stress is general during early somatic embryogenesis. They might play an important role in the activation and developments of longan somatic embryos. MW of most unknown proteins was lower than 30 kD, which were critical in regulation of somatic embryogenesis.4. Cloning of several embryogenic cultures associated protein genes at early stage during somatic embryogenesis of longan①The full-length cDNA sequence of longan embryogenic callus mitochondrial F1-ATPase beta subunit gene was 2099 bp. It contained a 1677-nucleotides-long open reading frame (ORF) which encoded protein of 588 amino acid residues. Transcription level of mitochondrial ATP synthaseβsubunit changed with development of somatic embryogenesis in longan and reached highest at GE stage, but its protein expression level remained constantly. The results showed the increase of its transcription level was good for stabilization of this protein, and could supply sufficient energy to the morphogenesis forming.②Two longan embryogenic callus triosephosphate isomerase (TPI) genes were cloned. The cDNA full-length of TPI I was 1084 bp and its ORF was 765 bp which started from the 73rd base and ended in the 837th base. It encoded 254 amino acids. The cDNA full-length of TPI II was 1113 bp and its ORF was 765 bp which started from the 157th base and ended in the 921st base. It encoded 254 amino acid residues. A pair of primers were designed according to the conserved sequence of these two TPI genes and used for real-time fluorescence quota PCR analysis. The results indicated their mRNA transcription level was low at FEC stage, then increased sharply at ECII stage and remained stable until CpECGE stage. The level decreased dramatically at GE stage and remained at a lower level from GE stage to cotyledonary stage. These implicated they had other function during somatic embryogenesis in addition to basal metabolism.③DlUP-3 gene was cloned and its cDNA full-length was 1681 bp. Its ORF was 1017 bp which started from the 391st base and ended in the 1407th base and encoded 338 amino acids. Its sequence was highly similar to that of castor-oil plant putative GTP-binding protein. Its transcript level was low at FEC stage and over expressed at GE stage. It might be a specific protein, which played an important role during GE stage forming in longan.④DlUP-4 gene was cloned which cDNA full-length was 1061 bp. Its ORF was 735 bp which started from the 1121st base and ended in the 846th base and encoded 244 amino acids. Its sequence was high similar to other plants and 60S ribosomal protein L7 of Arabidopsis thaliana. It specifically expressed during somatic embryogenesis in longan, and might involved in synthesis of somatic embryos related specific proteins.⑤DlUP-5 gene was cloned which cDNA full-length was 887 bp. Its ORF was 681 bp which started from the 7th base and ended in the 687th base and encoded 226 amino acids. Its sequence was highly similar to that of castor-oil plant putative Frigida. According to its variations of mRNA transcription levels, It might play a critical role in maintaining ability of somatic embryogenesis and a particular role in early somatic embryogenesis and maturation of somatic embryos.⑥Cloning of two unknown protein genes related to early somatic embryogenesis in longan. Conserved regions and 3 'end cDNA sequences of DlUP-6 and DlUP-7 were obtained. DlUP-6 gene and DlUP-7 gene were the two unknown protein genes in other plants. Furthermore, compared with the peptide sequences information from mass spectrometry, it was primarily proved that the two conserved regions and the two 3 'end cDNA sequences were the unknown proteins in the 2D maps.
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