Isolation And Functional Characterization Of Cytosolic Glyceraldehyde-3-phosphate Dehydrogenase Family Genes In Rice

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classical enzyme of the glycolysis pathway. Historically, GAPDH was commonly used as a model protein for the analysis of protein structure and enzyme mechanisms, or as an internal control for relative quantitation of gene expression. In higher plants, two distinct isoforms of GAPDHs exist, which exhibit specific cell compartmentalization and are encoded by distinct nuclear genes. The cytosolic GAPDH (GAPC) catalyses the oxidative phosphorylation of glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate by converting NAD+ into the highenergy electron carrier, NADH. The phosphorylating NADP+-specific GAPDH (GAPA/B) located in chloroplast stroma is involved in photosynthetic CO2 fixation. In mammals, GAPDH has been implicated in processes such as membrane fusion, microtubule bundling, nuclear RNA export, DNA repair, phosphotransferase activity, transcriptional regulation, signal transduction cascades and apoptosis. The gene structure and biochemical and functional properties of GAPDHs have been thoroughly investigated in mammals. However, only a few plant GAPDHs have been functionally characterized. Recently, numerous GAPC genes have been isolated from plants, most plant GAPC genes analyzed were induced in response to various stresses, such as anaerobic stress in maize and soybean, heat, anaerobiosis, or increased sucrose levels in Arabidopsis, and dehydration or ABA in Craterostigma plantagineum, suggesting that GAPC genes may function in the plant stress response.Rice is a major crop worldwide and a model for the functional genomic study of monocot species. In the present study, we isolated five genes that encode putative GAPC proteins in the rice genome, analyzed the sequences of OsGAPC genes, and investigated their expression profiles of this family in various organs and in leaf tissues exposed to various abiotic stresses. One of OsGAPC genes (OsGAPC3) was over-expressed in rice to evaluate its possible involvement in salt stress tolerance. Our results suggest that the OsGAPC gene family may play important roles in the response to environmental stresses. The main results are as follows:1. BLAST searches of the rice genome annotation databases at TIGR and KOME were performed, three GAPC genes were identified in the rice genome. Basic information about the OsGAPC genes is collected. OsGAPC1, OsGAPC2, and OsGAPC3 possess exons that are conserved in length and position, respectively. OsGAPCs family members have two typical domains:a NAD-binding domain and a C-terminal catalytic domain. The full-length amino acid sequences of rice GAPC proteins share 84-93% sequence identity and 88-97% similarity. All five OsGAPC sequences contain two cysteine residues.2. A total of three OsGAPC genes were cloned and constructed for over-expression, stably inherited homozygous lines were chosen.3. OsGAPC1, OsGAPC2, and OsGAPC3 had similar expression patterns in different organs, expression was highest in seedling roots and shoots, in booting leaves, and in flowers. Interestingly, all of the OsGAPC genes had little or no expression in booting culms. All of the OsGAPCs were induced by almost all stress factors. The high expression of these OsGAPC genes may correspond to their important and redundant roles in plant metabolism as a key enzyme of carbon metabolism. the expression of some OsGAPC genes was increased rapidly and abundantly during drought or heat treatment suggests that an acceleration in glycolysis is one of the first cellular responses to water deficit and heat shock, which have a marked effect on the respiratory capacity of mitochondria. 4. Three independent lines (OE-1, OE-4, OE-5) of transgenetic rice plants showed significantly increased tolerance to salt stress both during and after germination. Furthermore, the elevated stress tolerance of OsGAPC3-overexpressing plants coincides with the upregulation of several stress-responsive genes, including DREB2A, Lip9 and catA. In consistent, H2O2 was present in lower levels in OsGAPC3-overexpressing plants compared with WT under both normal and high salt conditions. The level of proline was the same in the transgenic rice and WT. We propose that OsGAPC3 plays an important role in the control of H2O2 over-accumulation in plants, which in turn enhances salt tolerance in transgenic plants.5. Gene silencing by amiRNAs was demonstrated for ZH11, amiRNAs of 3 single GAPC gene and the gene family were constructed for RNAi suppression. Our results show that amiRNAs can efficiently trigger gene silencing in a monocot crop. amiRNAs have the potential to specifically reduce the activity of the gene family and only one of the parental alleles.