Proteomic Analysis Of Rice Somatic Bud Regeneration And Gene Function Validation

Plant root and shoot development and their molecular mechanism is a hot area in development biology. The present study was attempted to identify the genes that are closely related to rice root and shoot development via in vitro individual organ specific induction, protein differential display and mass spectrum analysis. And then, to make clear the candidate genes'biological function and their effect to regulate rice root and shoot development via cDNA cloning, gene over-expression, and RNAi repression.The main results were summarized as followings:1. Shoot and root in vitro regeneration systems were successfully set upAn elite indica rice restorer line, MH63, was selected as starting material for callus induction. The medium for regeneration of individual shoot or root or whole plant were optimized by adjusting the concentration of plant hormones and other components. The calli were then inoculated onto these optimized media, respectively, for organ specific induction. The results showed that the calli induced on the shoot specific induction medium grows only shoot but no root, with the ratio up to 95.3%, while the calli induced on the root specific induction medium grows only root but no shoot, with the rate no lees than 71.0%. The higher rate up to 91.3% was also obtained for the whole plant induction. These results thus indicated that an in vitro organ specific induction system applicable for large scale protein sampling and subsequent 2-D electrophoresis has been successfully set up.2. Microscope observation of somatic root and shoot developmentThe data derived from the microscope observation indicated that somatic root and shoot differentiation is started from the several layers of cells on the callus surface. Four days after inoculation, the differentiation differences of the callus surface cells on the shoot and root specific induction media was becoming clear. At the same time, the differentiation progressed into inner cells; another four-day after that, the root meristem derived from the several layers of callus surface cells was becoming clear, while the shoot meristem derived from the several layers of callus inner cells began to develop. At the same time, the transport tissues were accompanying them to develop. Twelve days after inoculation, the differentiation of the different organs was further progressed; Up to sixteen days after inoculation, the shoot and root specific induction was finished. However, the whole plant differentiation at the moment was still continued, lagging behind about 3 to 5 days as compared with that of root and shoot.3. Proteomic analysis of somatic shoot and rootStarted from zero days, soluble proteins was extracted every four days from the differentiated calli at the different developing stages and used for protein differential express display. The numbers of 102 divergence protein spots were identified via PDQuest analysis. Among them, the sequences of 83 proteins contained in the 78 spots were determined using mass spectrum. Further analysis indicated that these proteins could be classified into 10 categories based on their function. The first four categories were function unknown (36%), protein destination/chaperor (15%), defense related (14%), and metabolism related (12%), accounting for 77% of total sequenced proteins.4. Molecular cloning of leaf midrib related gene and its functional analysisGene corresponding to protein spot 5108 with clear differences among samples and developmental stages in 2-D gel (the numbers were given by software PDQuset) were selected and cloned for further functional dissection. The results shows that the protein spot 5108 encode a 1-Cys-Proxiredoxin protein and plays an important roles in leaf midrib development. RNAi repression analysis indicates that once this gene's expression was repressed the clear cells in the middle of leaf midrib disappear so that the midrib developed abnormally and can not act to support leaf any more. The leaf blade thus droops down. In addition, the leaf base distorts so that the leaf back side turns up.5. Analysis of causes leads to sequence chimeras during cDNA cloningWhen clone two members of a closely related rice 33-kDa secretory protein gene family, we found existence of 20% sequence chimeras in the final products. These sequence chimeras generally switched from 5'-end of one member to 3'-end of another member, or vice versa. To make clear how these chimeras were produced, we systematically analyzed their occurrence causes. The data indicate that the mix template amplification is the major source leading to formation of these sequence chimeras. Their occurrences mechanism was confirmed mostly due to incomplete DNA amplification. While this incomplete DNA amplification was largely affected by PCR buffer additive and DNA polymerase species. In addition, our data also reveal that heteroduplex repairs by nick-dependent microbial DNA repair systems in the cDNA cloning step were found to produce chimeras, although it was not the major source under the present conditions. Based on these results, several simple strategies have been suggested to reduce the formation of the chimeras in the mixed-template amplification and subsequent cDNA cloning.