The Role Of OsPCF6and OsTCP21in Cold Stress Signaling Pathway From Rice

Cold stress is one of the most important limiting factors affecting rice production in temperate,adversely affecting the plant growth and development, can significantly constraint the spatial distribution and agricultural productivity. Cultivating new psychrotolerant varieties is the most effective way to develop agricultural yield of rice. Since cold stress response is a process that requires the participation of many genes, the effect of ectopic expression for a single functional protein to increase cold tolerance is very limited. Transcription factors play critical roles in the plants response to abiotic stress, single gene expression can start signaling network via transcriptional regulation of the downstream genes responsible for plant tolerance, so as to achieve the more significantly effect on improving crops resistance to stress. Therefore, screening for stress responsible transcription factors will provide more efficient and important gene resources for crops improvement.In this study, we firstly confined miR319detected in the microarrays as cold responsive miRNAs with real-time PCR. Then, over-expression of miR319was done with the Agrobacterium co-cultivation method to check their effect on cold response. After identifying miR319-OsPCF6/OsTCP21pairs by combining bioinformatics tools and microarray data analysis, we detected the expression of putative target gene in overexpression lines. We also investigated the roles of OsPCF6and OsTCP21in the plant’s response to cold stress by using overexpression and RNAi transgenic rice. Collectively, the results suggested that, as Osa-miR319targets, OsPCF6and OsTCP21negatively regulated plant cold tolerance, perhaps through mediating ROS generation or scavenging.The main results in this study are as follows:1. Isolation and sequence analysis of OsPCF6and OsTCP21from riceThe full length cDNA of the OsPCF6and OsTCP21genes were isolated by homologous cloning method. The full-length cDNA of OsPCF6is1854bp, the complete length of CDS is1074bp, encoding a357amino acid protein. The full-length cDNA of OsTCP21is2108bp, the complete length of CDS is1338bp, encoding a445amino acid protein.2. Verification of TCP genes in response to cold stress in riceWe used real time-PCR to analysis the expression of OsPCF6and OsTCP21genes under cold stress in wild type and overexpression Osa-miR319b plant. We found OsPCF6and OsTCP21were up-regulated at Ih and then significantly down-regulated within3h. 3. Subcellular localization of OsPCF6/OsTCP21proteinsThe GFP instantaneous expression system was used to analyze the transcription factors subcellular localization. The subcellular localization vector of OsPCF6and OsTCP21were constructed, and the GFP-fusion proteins were transiently expressed in onion epidermal cells via biolistic genetic transformation. The OsPCF6-GFP and OsTCP21-GFP fusion proteins were localized to the nucleus using a confocal laser scanning microscope, which suggested that OsPCF6and OsTCP21might act as transcriptional regulators to control signalling transduction.4. Transcriptional activation activity analysis of OsPCF6/OsTCP21The full-length OsPCF6and OsTCP21gene were cloned into pGBKT7vectors and introduced into yeast using the LiAc method. β-galactosidase activity indicated that OsPCF6could not activate gene expression, while OsTCP21could activate gene expression. These results suggest that OsPCF6may act as a repressor of gene expression, while OsTCP21may act as an activator.5. Functional analysis of OsPCF6/OsTCP21by transgenic lines in riceWe constructed OsPCF6and OsTCP21plant over-expression/RNAi vectors and accomplished the Agrobacterium mediated genetic transformation of rice callus. Through PCR, Southern blotting and Real time-PCR detection analysis, we obtained more than three transgenic lines for each gene. Then, we used quantitative real-time PCR to detect the OsPCF6and OsTCP21expression levels. The level of OsPCF6and OsTCP21were increased in overexpression transgenic lines, but level of miR319a had decreased comparing with the wild-type and overexpression Osa-miR319plant.6. The molecular mechanism of OsPCF6IOsTCP21in cold stress responseWe analyzed the cold-related gene expression levels in the OsPCF6and OsTCP21over-expression plants and wild-type. The CBF/DREB transcription factor could activate the expression of many COR genes. They can bind to the cold-and dehydration-responsive DNA regulatory elements (DRE), also termed C-repeats (CRTs), which is sufficient to induce transcription under cold stress. RNAi of OsPCF6and OsTCP21led to a greater accumulation of osmolytes, such as free proline, and DREB1/CBF proteins in rice, and suppressed the accumulation of ROS under conditions of cold stress through down-regulating OsPCF6and OsTCP21.