Characterization Of NAC Family Transcrition Factor OsNAP Conferring Abiotic Stress In Rice

Rice, a staple crop which provides dietary nutrition for over half of the world’spopulation. The extreme environments and genetic characteristics of plant often affectthe yield and quality seriously. Abiotic stress such as drought, high salinity and lowtemperature are among the major environmental factors which limit the productivityand growth potential of plant. Researchers from all over the world have made greatefforts in understanding the mechanisms of responses to abiotic stresses in rice. Plantmust adapt their growth and development to detrimental conditions by activating avariety of signaling pathways, which includes the interplay of stress response genes.While individual genes have been reported to improve the stress tolerance of crop, thenumber of environment in which these genes are required transcription factors (TFS).NAC transcription factor is a newly discovered plant specific transcription factorin recent years. It has a variety of biological functions, which expression is induced bybiotic stresses and abiotic stresses. It modulates plant growth, development and stresstolerance. There are117members of NAC family in rice genome, many of them haveproven to be involved in stress response pathways AtNAP gene has been shown topostpone leaf senescence in Arabidopsis thaliana, but its function related to plantabiotic stress about the homolog gene in rice remains to be addressed.This study cloned OsNAP (Os03g0327800),a homologous gene of AtNAP(Atlg69490),which associated with aging in Arabidopsis thaliana, from rice (Oryzasativa.)genome through bioinformatics analysis. For further study of OsNAP function,we construct pCAMBIA1304-35S: OsNAP vector and pCAMBIA1304-35S: OsNAP-SRDX expression vector. T3homozygote strains were evaluated for abiotic stress. The results showed as follows:1. Cloning OsNAP.Using homology-based cloning method, RT-PCR technique andNCBI (the national biological information centre) website, OsNAPwere cloned fromrice genomic DNA. The open reading frame (ORF) of OsNAP was1179bp in length,which encoded a392amino acids sequence with isoelectric point of8.55andmolecular mass of42.195ku.2. OsNAP localizes to the Nucleus. Observation of the green fluorescence by stableexpression of the OsNAP-GFP fusion protein in rice revealed the specific nuclearlocalization of OsNAP, implying a role of OsNAP as a transcription factor.3. Expression profile analysis. The expression of the OsNAP under exogenoushormones (6-BA,GA, ABA,etc.) were examined by qRT-PCR. the result showed thatOsNAP was strongly induced by ABA, but not6-BAand GA;Similary, the expressionof OsNAP gene was strongly induced by NaCl，PEG and H2O2and slightly inducedby low temperature; qRT-PCR analysis revealed that OsNAP is expressed in varioustissues, with a relatively higher expression in leaves,especially in the old leaves withthe maximum level.4. Stress tolerance of35S:OsNAP Plants. Transgenic rice constitutively expressingOsNAP and OsNAP-SRDX were also generated. Four T3overexpressionhomozygote lines (OX1OX10, OX11, OX17), and three high expression SRDX lines(SR4, SR6, SR7) were selected for stress tolerance analysis. The35S:OsNAP plantsshowed significantly enhanced salt and drought stress tolerance.5. Expression of stress-related genes in the OsNAP transgenic lines were verified: wevalidated the expression of genes related to abotic stress from transgenic lines(OX11)and wild type Nipponbare through real-time PCR. It was found that the expression ofkey gene such as PP2C,Os01g0846300, ABI2, DREB1A,MYB2, SalT, and AP57in thetransgenic lines is much more than that in the wild type. It shows that OsNAP mayregulate a large number of stress-related genes, many of them predicted to bedownstream genes of ABA depended stress response signaling pathways.