| Comparing to C3plants, C4plants have higher photosynthesis efficiency.and introducing the C4core cycle related enzymes into C3plants by genetic engineering was considered as one of the effective efficient strategy to increase its photosynthesis efficiency. However, the potential photosynthetic regulation mechanisms, such as the photosynthetic apparatus formation and the differentiation and positioning of mesophyll chloroplast and bundle sheath chloroplast, are remaining unknown. Many species in Eleochairs use different photosynthetic models in different environments and this feature make them to be an ideal system in C4regulatory research. In this study, SSH and NGS were used to identify the C3/C4differentially expressed genes in Eleocharis baldwinii. The main aims are to analysis the C3/C4metabolic differentiation and obtain candidate genes for C4regulatory research. The main results are as follow:1. A terrestrial up regulated SSH library was constructed using terrestrial cDNA as the tester and submerged cDNA as the driver.64terrestrial up regulated ESTs were identified by the total cDNA probes of terrestrial and submerged. After bioinformatics assay,56unigenes were confirmed and52of them have GO annotations. These genes mainly involved in C4core cycle, metabolic transportation, photosystem and regulatory genes, such as transcription factor. However, the information that SSH supplied was very limited;2. Three RNA samples including terrestrial, ABA induced and submerged were deeply sequenced by NGS (Next Generation Sequencing). After clean reads assembly, we totally obtained61747unigenes with an average length of471bp and the length distributed between200bp to3000bp. At last,24204DE (Differentially Expressed) genes were identified;3. The clustering results of DE genes showed the gene expression profiles in ABA induced sample was more similar to those in terrestrial. At the same time, the DE genes can be divided into9clusters according the different expression profiles. The cluster I, III and VI represented the most of C3/C4differentiation and the genes in cluster IX might relate to the environment responding;4. After gene functional assay, we found:1) The C4core cycle related enzymes and a huge of metabolic reporters were up regulated in terrestrial and ABA induced samples and these transporter may contribute to C4photosynthesis in both metabolism fluxes and developmental regulatory;2) The genes related to Calvin cycle, the citrate cycle and glycolysis were also up regulated in C4culms and their roles in C4photosynthesis may relate to the increasing photosynthetic efficiency and supply carbon skeleton for CO2fixation;3) The down regulated ribosome complex related genes suggested a less protein synthesis and a higher N use efficiency in C4culms;4) The different expression profiles of light harvesting and C2cycle related genes between terrestrial and under water (both submerged and ABA induced) may relate to the environment responding and not directly relate to C4photosynthesis, such as genes of chlorophyll binding protein, biosynthesis of chlorophyll and carotenoids and phytochrome responding genes;5) The different expression profiles of ABA biosynthesis and catabolism related genes in terrestrial and ABA induced sample indicated that an increasing endogenous ABA biosynthesis and additional exogenous ABA complementation were two different strategies to maintain endogenous ABA homeostasis in C4photosynthetic regulatory pathway in E. baldwinii.5.1205C3/C4differentially accumulated regulation genes were identified including830transcriptional factors and375protein kinases. The Real-time results suggested that some regulation genes were co-expressed with the development procedure of photosynthetic apparatus and chloroplast accumulation and differentiation. These kind of genes may involve in the development controlling of photosynthetic apparatus and will be served as the candidate genes in the gene functional validation;6. In this study, some genes related to epigenetic modification were also differentially expressed in C3/C4samples. The MSRE-PCR results of PPDK genomic DNA showed there were indeed different methylation levels in terrestrial and submerged environments.
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