Isolation And Functional Analysis Of Tomato SINAM1 Transcription Factor

Cold stress, including chilling stress?< 10°C? and frozen stress?< 0°C?, is a significant factor limiting geographical distribution of plants and agricultural production of crops. Chilling stress can directly affect physiological metabolism and stunt the development of plants. Chilling stress reduces photosynthetic rate of plants and the synthesis, allocation and transport of photosynthetic products are all influenced seriously. Chilling stress even induces secondary dehydrated and ostimic stress by inhibiting water uptake. Moreover, reactive oxygen species?ROS? accumulation are induced by chilling stress, which could destroy the structure of biomacromolecule and cytomembrane. Solanum lycopersicum originates from subtropical area and can't be acclimated by chilling stress. Thus, low temperature is a crucial factor limiting production of tomato.NACs are plant-specific transcription factor family and most of these TFs are inducible transcription factors. The functions of NAC proteins are various, including plant growth and development, abiotic and biotic stress response. Bioinformatics analysis showed that there are as many as 102 NAC transcription factors in tomao. However, the functions of many NACs in tomato are still unknown. A novel NAC transcription factor named SlNAM1 was isolated and characterized. Then SlNAM1 overexpression tobaccos were used for further research. In this paper, we analyzed the germination rate, growth performance, accumulation of ROS, damage extent of biological membrane, osmoprotectants contents and expression levels of downstream stress-related genes of transgenic plants under chilling stress. The main results are as follows:?1? A pair of specific primers was designed based on the m RNA sequence?Gen Bank accession number NM₀01247290.1?. Then PCR was performed for isolation of SlNAM1 using the primers. The full-length of SlNAM1 is 1218 bp with a 891 bp open reading frame, which encodes 296 amino acids.?2? Transient and stable expression assay of p35S-SlNAM1-GFP fusion protein in tobacco leaves were performed. The green fluorescence was observed only in nucleus, indicating that SlNAM1 is located in nucleus. Transcriptional activation activity assay of SlNAM1 was performed through yeast two-hybrid system and C-terminal of SlNAM1 exhibited transcriptional activation activity. These results suggested that SlNAM1 functions as transcription factor.?3? The expression pattern of SlNAM1 was analyzed through q RT-PCR. The expression levels of SlNAM1 were induced by 4°C, PEG, Na Cl, ABA and JA treatments. Moreover, the tissue specific expression of SlNAM1 showed that the expression level of SlNAM1 was highest in leaves.?4? The recombinant expression vector p BI121-SlNAM1 was constructed and transformed into tobacco using a method of Agrobacterium through leaf disc transformation. The transgenic tobacco was identified by PCR and q RT-PCR. Besides, OE-7, OE-10, OE-12 were selected for further research.?5? The growth status of transgenic tobacco was consistant with WT tobacco under normal condition. However, the transgnic tobacco showed higher germination rate, lower content of wilting and higher net photosynthetic rate compared to WT under chilling stress.?6? The accumulation of ROS was increased under chilling stress. However, the transgenic tobacco exhibited lower ROS accumulation and higher SOD and APX activities than that in WT tobacco. The expression levels of key genes involved in sythesis of antioxidant enzyme was higher than WT plants, indicating that the transgenic tobacco maintained higher antioxidant enzyme activies by increasing the synthesis of antioxidant enzyme through upregulating the related genes. Further more, MDA contents and REL of transgenic tobacco were lower than that in WT plants, suggesting that the transgenic tobacco with SlNAM1 alleviated the oxidated damage to some content.?7? Some stress-related genes, such as Nt P5 CS, Nt DREB1 and Nt ERD10 s, are up-regulated by chilling stress and the transcripts of transgenic tobacco were higher than that in WT plants. Meanwhile, the contents of osmolytes, such as proline, soluble sugar, were increased and the increases were much higher in transgenic plants than that in WT plants, which is conducive to maintaining cell osmotic potential, alleviating osmotic stress induced by chilling stress, maintaining the stability of cell membrane, and improving the chilling stress tolerance.