| Litchi (Litchi chinensis) was one of the tropical precious fruit trees in southern China, which had been cultivated more than 2000 years in China; meanwhile, litchi which was also an important-representative landscape tree of the tropical and southern-subtropical scenery in southern China and was widely used in urban landscaping and scenic areas. Ancient and famous trees were usually an unique landscape in the Landscape Architecture, were one of the best sceces in dynasties cemetery and historical sites, one of the important natural and cultural landscape in the city. The gene mapping of the ancient trees usually had its particularities, it usually had longevity genes, resistance genes, and other valuable genes; therefore, it was the most valuable germplasm material in plant genetic improvement. Ancient litchi trees in Fuzhou were rich in germplasm resources, and played an important role in the history of the litchi cultivation. In view of this, genetic diversity in Fuzhou ancient litchi trees was first analyzed by the RAPD molecular marker, then the in vitro restricting conservation of core germplasm in Fuzhou ancient litchi trees were conducted, and further the mechanism of in vitro conservation in Fuzhou ancient litchi trees was analyzed by the methods of molecular biology and proteomics. The main results were as follows:1. RAPD analysis of genetic diversity in Fuzhou ancient litchi trees In this experiment, the genetic diversity of 66 accessions (2 of them were said to be the Offspring of ancient litchi tree"shibaniang"from the West Lake in Fuzhou City)of ancient litchi trees was evaluated by using RAPD (Random amplified microsatellite polymorphism). The results showed that 232 brands were amplified by 16 random primers, among which 216 were polymorphic; the ratio of polymorphic brands was 93.10%, which suggested that 66 accessions of ancient litchi trees have abundant polymorphism. According to the polymorphism of the amplified bands, a dendrogram showing genetic relationships among ancient litchi trees was constructed by UPGMA, and the genetic distances varied from 0.000 to 1.000. When D was 0.909,66 accessions of ancient litchi trees could be divided into 3 groups; When D was 0.746, they could be divided into 9 groups; When D was 0.576, they could be divided into eighteen groups, while the ancient litchi trees in Fuzhou area could be divided into seventeen groups, the other group contained 2 litchi trees which were said to be the Offspring of ancient litchi tree"Shibaniang"in xihu Fuzou. Ancient litchi trees at the same plot almost could be divided into one group, though some individual which had a distant relationship were in the different group, which showed that there was a genetic variation among ancient litchi trees in Fuzhou area. According to the representation of RAPD bands in ancient litchi trees, and considering other factors such as tree ages and famousness, 17 ancient litchi trees were firstly determined to be the core germplasm of ancient litchi trees in Fuzhou area.2. The in vitro restricting conservation and somatic embryogenesis of germplasm in Fuzhou ancient litchi treesThe EC(emberyogenic callus)of 17 accessions of core germplasm in ancient litchi trees had been induced by anther culture, and the best way for in vitro restricting conservation of embryogenic calli in ancient litchi trees was as follows: the inoculation amount of embryogenic calli was 50 mg per bottles, then was divided into 3 clusters and cultured on the MS medium with 2,4-D1.0 m g?L-1, mannitol 2%, sucrose 20 g?L-1, agar 8 g?L-1 ,with the pH value of 5.8, at 20℃in darkness. By the conservation method, the longest subculture time could be prolonged to 125 days. The embryogenic calli had a strong capacity of somatic embryogenesis after conservation. The genetic variation in conserved embryogenic calli was detected be RAPD molecular marker, the results showed that the variation was only 7.70% after the in vitro restricting conservation.3. Study on the activities of glutathione peroxidase (GPX) and cloning of GPX from EC in the ancient litchi treeThe ECS under the conventional conservation and the best in vitro restricting conservation were used as the materials for the studying the changing tendency of glutathione peroxidase (GPX) activities in the embryogenic callus during conservation. The results showed that GPX had a regular expression in the embryogenic callus during the conventional conservation and the best in vitro restricting conservation, and they both had two peaks, the results indicated that GPX in the embryogenic callus was closely related to its in vitro conservation. The appearance of the first peak of GPX activity was related to the mass propagation of embryogenic callus, which was the sign of the coming growth peak. The appearance of the second peak of GPX activity was the sign of the entering into the aging period. But the appearance time of the two peaks of GPX activities in the embryogenic callus under the best in vitro restricting conservation was much later than the compared (under the conventional conservation).The EC induced from the anther of"Songli"in Xichansi Temple was used as the material for the cloning of the full-length cDNA and DNA of GPX gene. The ORF of the GPX gene from the ancient litchi tree EC was 507 bp (The registered No. was FJ172343); and the full-length DNA of ancient litchi tree EC GPX gene was 1809 bp(The registered No. was FJ465145), which was consisted of 5 exons and 4 introns. Meanwhile, the physicochemical properties, hydrophilic and hydrophobic characteristics, secondary structure, three-dimensional structure, transmembrane structure, domain and functional domain, signal peptide, phosphorylation sites, motif, subcellular localization, and molecular evolution of the GPX protein was predicted by the analysis of the amino sequences using the bioinformatics softwares.4. Proteomics research during in vitro conservation of the embryogenic callus in the ancient litchi treeProteomics research in the process (for 15 d, 20 d, 30 d) of the conventional conservation of the embryogenic callus in the ancient litchi tree by immobilized pH gradients two-dimensional electrophoresis and biological mass spectrometry (peptide mass fingerprinting and tandem mass spectrometry), and the difference between embryogenic calli for 30 d in the ancient litchi tree under the conventional conservation and the best in vitro restricting conservation was compared.(1) The changes of protein components, MW and pI in the process of in vitro conservation of the embryogenic callus in the ancient litchi treeThe changes of the protein component, MW and pI value in the process of the conventional conservation of the embryogenic callus in the ancient litchi tree (for 15 d, 20 d, 30 d) and embryogenic callus for 30 d under the best in vitro restricting conservation showed that:①with the culture time increased, the change of the protein number was an upward trend after the first drop, the number of the corresponding proteins was 1311, 1256, 1441 in the process of the conventional conservation of the embryogenic callus (for 15 d, 20 d, 30 d), respectively; the protein number under the best in vitro restricting conservation was less than the conventional conservation, only 1023, which showed that more proteins were synthesized both in the rapid growth period and close to the aging period, while less proteins were synthesized under the best in vitro restricting conservation, with a state of "dormancy".②with the culture time increased, the proportion of small molecular weight (<15KD) proteins was gradually reduced; while it was increased significantly under the best in vitro restricting conservation, which might be that the small molecular weight proteins had the function of maintaining normal growth of the embryogenic callus.③with the culture time increased, the proportion of proteins with pI values between 4 and 5 gradually increased, the proportion of proteins with pI values between 5 and 6 had smaller changes and the proportion of proteins with pI values between 6 and 7 gradually reduced; under the best in vitro restricting conservation, the proportion of proteins with pI values between 5 and 7 gradually increased, in particularly, the proportion of proteins with pI values between 5 and 6 increased significantly, while the proportion of proteins with pI values between 4 and 5 was smaller, which conjectured that pI values of the proteins which had the functions of maintaining normal division and proliferation of the embryogenic callus were mostly between 5 and 7, the pI values of the proteins which had the functions of the aging or anti-aging/oxidation were mostly between 4 and 5.(2) The MS identification and function analysis of the associated proteins in the process of in vitro conservation of the embryogenic callus in the ancient litchi treeThe in vitro conservation-associated proteins separated from the two-dimensional electrophoresis were identified by the method of PMF or tandem mass spectrometry, 39 proteins were successfully identificated (p<0.05). The successfully identified proteins were divided into eight functional groups related to the amino acid metabolism (1 protein), lipid metabolism (2 proteins), nucleic acid metabolism (2 proteins), protein metabolism and repair (15 proteins), cell composition and signal transduction (2 proteins), energy and carbon metabolism (4 proteins), stress response and redox (8 proteins), unknown protein (5 proteins ).The function analysis of successfully identified proteins indicated that the expression of energy and carbon metabolism associated proteins of the embryogenic callus in the process of the conventional conservation was first reduced and then tended to increase; the expression of the signal transduction, lipid metabolism and amino acid metabolism associated proteins was gradually downtrend; the expression of the stress response and redox, cell composition, nucleic acid metabolism, protein metabolism and repair associated proteins was stable.The expression of the stress response and redox, nucleic acid metabolism, amino acid metabolism, lipid metabolism and protein synthesis associated proteins of embryogenic calli for 30 d under the conventional conservation was stronger than that under the best in vitro restricting conservation; a few differences in the expression of protein degradation, energy and carbon metabolism, cell composition and protein repair associated proteins between the conventional conservation and the best in vitro restricting conservation; the expression of signal transduction associated proteins under the best in vitro restricting conservation was higher than that under the conventional conservation. |
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