Functional Analysis Of Potato Transcription Factor StNAC1 Under Salt Stress

Salinization of farmland is a fast-growing problem worldwide due to long-term unreasonable irrigation.According to statistics,salt stress reduces at least 20%crop yields in recent years.Potato（Solanum tuberosum L.）is the fourth largest food crop in the world.Genetic resources exploitation and the underlying mechanism resolution.This crop is crucially important for the development of salt-tolerant varieties and and global food security.The NAC transcription factors family is a type of plant-specific transcription factor and one of the largest transcription factor family.Its members are widely involved in the regulation of various stress responses in plants,including salt stress.However,the molecular mechanism of potato NAC transcription factors in response to salt stress is rarely reported.In this study,the potato St NAC1 gene was cloned,its tissue expression and salt stress induced expression pattern were analyzed.We overexpressed St NAC1 in tobacco and characterized the phenotype and multiple physiological indicators of the transgenic plants under salt stres.The main research results have been showed as follows:（1）Molecular bioinformatics analysis.Amino acid sequence alignment and protein structure prediction showed that St NAC1 protein share a sequence similarity of96.35%with tomato（Solanum lycopersicum L.）Sl NAC1,and these two NAC1 both belong to ATAF subfamily.Therefore,it was speculated that St NAC1 may play important role in salt tolerance,same as Sl NAC1.The molecular weight of St NAC1 is34.86 k Da,which is a stable hydrophilic protein.The 3D spatial structure model analysis of the interaction between St NAC1 protein and double-stranded DNA showeds that St NAC1 exists as a dimer.This protein contains a highly conserved NAM domain for DNA binding and a variable transcription activation domain at the C terminal.These results indicates that St NAC1 has the functions of DNA binding and transcription factor.（2）Tissue expression and salt stress induced expression analysis of St NAC1.St NAC1 gene was consistently expressed in leaves,stems and roots,but the expression level in leaves was significantly higher than that in stems and roots.At the same time,the expression of St NAC1 gene in leaves,stems and roots were induced by Na Cl.After treatment with 150 m M Na Cl,the expression of St NAC1 gene increased to the highest level at 3 h.（3）Construction of St NAC1 transgenic tobacco plants and expression analysis.The St NAC1 gene was cloned and combined with p BI121,p BI121-Ca MV35S::St NAC1-GFP expression vector was constructed and transformed into tobacco,and 37homozygous transgenic lines were identified.RT-q PCR analysis showed that the transcription level of St NAC1 in the homozygous transgenic line was significantly higher than wild-type,while no difference was observed for the endogenous Nb NAC1.These results suggested that the St NAC1 gene could be transcribed normally in tobacco,and the overexpression of the St NAC1 gene does not affect the expression of the endogenous gene Nb NAC1.（4）Phenotype analysis and physiological detection of St NAC1 transgenic tobacco plants.The germination rate of transgenic tobacco seeds was significantly higher than wild-type under 100 m M Na Cl stress.The green leaf rate and fresh weight of transgenic plants were significantly higher than wild-type plants under 150 m M Na Cl stress.The content of chlorophyll and proline were also significantly higher than wild-type.After3 h of 150 m M Na Cl stress,the DAB（H₂O₂）and NBT（O₂.^-）histochemical staining degree of transgenic plants seedlings was significantly weaker than wild-type tobacco plants.These results indicated that overexpression of the St NAC1 gene significantly enhanced the salt tolerance of transgenic tobacco plants.In summary,our study cloned and identified a new salt-tolerant gene St NAC1,which was induced by salt stress.Overexpression of St NAC1 in tobacco could reduce the accumulation of reactive oxygen species（ROS）induced by salt stress,increase the proline content,and significantly enhance the salt tolerance.Taken together,our work not only laids the foundation for the analysis of the molecular mechanism of potato salt stress response,but also provides a novel idea and candidate gene for improving salt-stress tolerance in potato.