| Nature rubber (NR) from Hevea brasiliensis(Hevea hereafter) is a significantly industrial and strategic raw material. The NR self-sufficiency rate of our country is very low, and the actually cultivated area of Hevea nearly reached its extreme. Therefore, substantially increasing the per unit yield of Hevea is the unique and credible way for our country. In regularly tapped Hevea trees, latex regeneration is the main metabolic activity occured in the laticifers cells, and consists of one of the main yield-limiting factors for rubber productivity. Latex regeneratin involves a complex network of gene expression and regulation, and the resultant physiological and biochemical process. High-throughput transcriptome profiling will lay a good foundation for the cloning of key genes and invetigating the mechanisms underlying the regulation of latex regeneration. Also, it will help identifying the important target genes used for directional molecular breeding.The effect of tapping on the activation of latex production was especially conspicuous in virgin trees of Hevea. This property makes virgin Hevea trees an ideal model to unravel the mechanisms underlying the regulation of latex regeneration. Here, for the first time the dynamic change of latex transcriptome was analyzed by high-troughput means for the first several tappings after opening of virgin Hevea trees. The main results are as follows:1. An in-depth latex transcriptome analysis after-opening performed for the first time in virgin rubber treesUsing a silver-staining cDNA-AFLP technology modified in our lab, with all the 128 selective primer combinations of Apo I IMse I enzyme restriction system, about 9000 transcript-derived fragments (TDFs) were identified in the latex for the first five tappings.With reference to the TDF levels in the first tapping, a total of 651 differentially expressed (DE) TDFs were cloned, sequenced and identified by homology searching. With the combination of the localized EST database in our lab, the DE-TDFs with the same expression pattern and functional annotation were integrated, and finally 505 non-redundant DE-TDFs were acquired,217 of which were classified as up-regulated,180 as down-regulated, and 108 as bell-regulated.366 (72.5%) DE-TDFs had homology to genes with known functions, whereas 17.6%(89) and 21.6%(109) belonged to either unclassified proteins or predicted proteins, and the remaining 9.9%(50) with no hit. Genes with known functions were classified into 11 functional categories, including transcription and protein synthesis, disease and defence responses, transporters and intracellular traffic, signal transduction, protein destination and storage, primary metabolism, energy, cell growth and division, cell structure, secondary metabolism and rubber biosynthesis.2. Multiple latex-regeneration raited genes screened and several key metabolic pathways govening latex regeneration proposedDuring the first five tappings after opening in virgin rubber tree, laticifer cells exhibited the phenomena of a strengthed RNA and protein synthesis, an accelearated protein turnover and intracellular transportation, and an activated signaling pathways and defense responses. The results indicated that tapping induced an obvious sink and wounding effect, in which multiple genes with different functional categories were involved.A number of DE-TDFs were isolated in the functional categories of sucrose transport. sucrose catabolism, glycolysis, tricarboxylic acid cycle and pentosephosphate pathways, most of which were up-regulated by tapping. Strikingly, the DE-TDFs of 9 rubber biosynthesis pathway genes were all up-regulated by tapping. The results suggest that the regulation of sucrose supply, sugar metabolism and rubber biosynthesis are the major metabolic activity in the laticifers cells after opening of virgin rubber trees, and it also constitutes the main reasons of strengthened latex regeneration with tapping.3. cDNA-AFLP technology being an reliable means for screening expressed genes in HeveaThe expression patterns of about 1/10 randomly selected DE-TDFs were confirmed by semiquantitative RT-PCR,81.25% of which consistent with the original expression patterns by cDNA-AFLP analysis, indicating this screening work was high reliablity. Meanwhile, the expression patterns of most of the 37 latex-regeneration related DE-TDFs were further confirmed by real-time RT-PCR.This results presented here not only help futher globally screening of the key genes involved in the regulation network of latex regeneration, but preliminarily addressed several key metabolic pathways determining latex regeneration, and provides the basis for futher revealing the molecular mechanisms of latex regeneration. |
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