| In Hevea brasiliensis, rubber biosynthesis and latex regeneration occur in specialized cells-the laticifers. Most of the enzymes and proteins implicated in latex regeneration are located in the cytoplasmic serum of the laticifers, i.e. the C-serum. Tapping stimulates latex regeneration and latex flow, which is conspicuous in the first several tappings after re-opeing in the rubber trees after a winter resting period. The simulated laticiferous metabolism and yield increase arise from the regulation of latex-regeneration related genes, which will be subsequently reflected in the changes in protein expression.In this paper, comparative proteome approaches were used to screen and identify the candidate latex-regneration related proteins, and the genes that encoded some candidate proteins were examined for their expression patterns in the latex with the tapping of re-opened rubber trees. The major results are as follows:(1) Proteins were extracted from the C-serum by different methods, and examined for their suitability in 2D-PAGE. TCA/Acetone precipitation followed by washing with eaqual volumes of alocohol and aether revealed to be a suitable method and the proteins were well separated by 2D-PAGE, and in each gel 1000-1200 discernable protein spots could be obtained by silver staining, and 700-1000 spots by coomassie blue staining.(2) Changes in latex yields and physiological characters demonstrated that tapping apparently stimulated the capacity of laticiferous metabolism and latex regeneration in the first several tappings of re-opened rubber tree. Therefore, the latex in the first several tappings was good material for investigating the regulation of latex regeneration.(3) With reference to the first tapping, the dynamic change of proteome in the C-serum was determined for the first five tappings. The expression patterns for the differentially expressed (DE) protein spots were classified into four types:up-regulation, down-regulation, bell-shape regulation, and inversed bell-shape regulation (i-bell regulation). A total of 117 DE protein spots were isolated and identified by MALDI-TOF or MALDI-TOF/TOF mass spectrometry, and 89 and 57 spots were successfully identified by MALDI-TOF and MALDI-TOF/TOF, respectively.(4) According to functional classification,23 protein spots belonged to the category of stress and defense response,18 to primary metabolism and energy,12 to cell growth, division and structure,9 to transcription, translation and protein fate,8 to rubber biosynthesis,4 to secondary metabolism,4 to signal transduction, and 11 to unknown proteins.(5) Regarding the expression patterns, the figure of the up-regulation protein spots apparently exceeds that of the down-regulation ones, but similar to that of the bell-and i-bell ones. In most functional categories, the DE protein spots showed multiple expression patterns, but all the 4 DE protein spots in signal transduction were up-regulated by tapping, and in the stress and defense category, most DE protein spots were showed a pattern of bell-or i-bell-regulation by tapping.(6) Some DE spots were not identified by MS searching in the public database, but had good matches to contigs when searching against the local latex EST library. The full-length cDNAs of six genes corresponding to the relevant contigs were cloned by RT-PCR and RACE technologies, including a glucose and ribitol dehydrogenase, a caffeic acid methyitransferase. an alpha-tubulin, an allergenic isoflavone reductase-like protein, a late embryogenesis abundant family protein and a cysteine proteinase inhibitor.(7) A total of 23 DE-proteins were selected for investigating the regulation of tapping on the expression of the genes with the same protein annotations. Primers were designed based on contig sequences in the local EST library, and real-time RT-PCR were employed to characterize the expression of 26 genes in the first 8 tappings in re-opened rubber trees. The results showed that expression patterns were not always consistent in the levels of protein and mRNA transcripts.The results presented in this paper have layed a good baisis for further unraveling the mechanisms underlying the regulation of latex regeneration, and for identifying the key genes implicated in latex regeration. |
PDF Full Text Request