Expression and Stress Tolerance Analysis of NAC24 from Tamarix hispida

Objective: The NAC transcription factors are one of the largest families of plant-specific transcription factors, widely involved in plant growth and development process, and it plays a crucial role in the process responding to salt, drought and many other abiotic stresses. In present study, we cloned a NAC transcription factors gene from halophyte woody plant Tamarix hispida, to study its salt tolerance and drought resistance function in order to lay a theoretical foundation for studying the molecular mechanism of resistance to woody plant NAC transcription factors. Method: A NAC transcription factor gene was isolated from NaHCO3 stress transcriptome database of T. hispida, and it was named as ThNAC24 (GenBank number: KF031949). Bioinformatics tools were used to perform multiple sequence alignment and phylogenetic analysis of NAC proteins from nine other species and 105 NAC proteins in Arabidopsis thaliana were analyzed. The T. hispida samples were treated with 300 mmol•L-1 NaCl and 400 mmol•L-1 Mannitol respectively, and the root and leaf tissues were taken at 6, 12, 24 and 48 h after the stress. The real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to analyze the expression of ThNAC24 gene under salt or drought stress in different time and tissues. It was initially identified whether it responded to salt or drought stress. To further study the stress resistance function of ThNAC24, overexpression (pROKⅡ-ThNAC24) and inhibitory expression vectors (pFGC5941-ThNAC24) were constructed. Overexpression (OE), inhibitory expression (IE) and control T. hispida plants were obtained by Agrobacterium-mediated high-efficient transient transformation system. The DAB, NBT staining, POD activity, SOD activity, electrolyte leakage, water loss rate, and MDA content under salt and drought stress were measured and compared with the transgenic T. hispida to identify the salt tolerance and drought resistance of the ThNAC24 gene. Result: The open reading frame of ThNAC24 gene is 1 023 bp, encoding 340 amino acids. Multiple sequence alignments results show that the amino acid sequence of ThNAC24 is relatively high in the N-terminal and has the sequence characteristics of NAC family. Phylogenetic tree analysis showed that ThNAC24 was closely related to ANAC103 and ANAC082. The result of qRT-PCR showed that the expression of ThNAC24 gene was up-regulated under salt or drought stress. The expression level of ThNAC24 was the highest at 12 h in root tissue, while it was the highest at 24 h in leaf tissue; Under drought stress, the expression level was the highest at 6 h in root tissue, while it was the highest at 12 h in leaf tissue. The ThNAC24 gene was expressed in roots and leaves of T. hispida and responded to salt and drought stress. Overexpression of the ThNAC24 significantly reduced the contents of H2O2 and superoxide anion, enhanced the POD and SOD activities in T. hispida, thus decreased ROS accumulation. Overexpression of the ThNAC24 gene can reduce the electrolyte leakage, water loss rate and MDA accumulation of T. hispida under stress, thus protecting the integrity of cell membrane structure. Conclusion: The ThNAC24 gene can respond to salt and drought stress. Overexpression of the ThNAC24 gene significantly enhances POD and SOD activities to improve ROS scavenging, reduces cell damage or death of T. hispida and improves its salt tolerance and drought resistance. © 2019, Editorial Department of Scientia Silvae Sinicae. All right reserved.