The Isolation And Functional Analysis Of NAC Transcription Factors In Peanut (Arachis Hypogaea L.)

Peanut is an important oil and cash crop. China is one of the vital peanut-cultivated countries in the world. Peanut distributes mainly in arid and semiarid region. Therefore, drought significantly affects the yield and quality of peanut. It is reported that 70% of peanut area in China are in various degree of drought and the average decrease in yield was more than 20%. Therefore, it is essential to resolve the negative effect of drought stress to peanut production.Plant is often exposed to biotic and abiotic stresses of varying degrees in growth process which affected plant growth and development badly and cause huge negative effect to agriculture. Plant has established some suitable and resistance mechanisms in the long evolution to reduce the damage by adjusting physiological and metabolic pathway. One of the important mechanisms is to induce the expression of stress response genes by regulating the stress signal transduction, and transcription factors have drawn more and more attention owing to their ability for regulating of many stress response factors.NAC transcription factors are the new type transcription regulatory factors which are unique to plants. They play an important role in regulation of plant growth and development, hormone level development, responses to stresses, and crops yield and quality. NAC transcription factors contain a NAC domain in the conserved N-terminal, which may function as DNA-binding region, while the various C-terminal serves as transcriptional activation region.We isolated two NAC-like genes from the cDNA library of different stages seeds of the export peanut cultivar'luhua14'by RT-PCR and named as AhNAC1 (GenBank accession number EU669863)and AhNAC2. Sequence analysis shows that the AhNAC1 cDNA has a length of 1453 bp which contains a single open reading frame(ORF) of 912 bp encoding a protein of 303 amino acids with an estimated molecular mass of 34.1 kD and an isoelectric point of 7.6. While the AhNAC2 cDNA is 1573 bp and contains an ORF of 1050 bp. It encodes a protein of 349 amino acids with an estimated molecular mass of 39.1 kD and an isoelectric point of 7.66. Compared to other NAM members, AhNAC1 and AhNAC2 belong to ATAF subfamily. We supposed that AhNAC1 may play a role in the seed development and AhNAC2 may be involved in drought response pathways.In this study, we characterized the AhNAC2 expression in response to drought, salinity, low temperature and ABA by semiquantitative RT-PCR analysis. The results indicated that the AhNAC2 expression was strongly induced by dehydration, and varied along with the drought treatment hours. However, exogenous ABA treatment didn't change obviously the expression of AhNAC2.Yeast transactivation analysis, domain deletion andβ-galactosidase filter assays showed that the transactivation activity of AhNAC2 was conferred by its C-terminal. Transient expression analysis in onion epidermal cells by genegun indicated AhNAC2-GFP fuse protein was specially localized in nuclear. All the results indicate that AhNAC2 is a transcription factor.Overexpression vector was constructed to examine the function of AhNAC2. By using Agrobacterium mediated leaf-disk tobacco transformation, the recombinant was transformed to Nicotiana tabacum L. The transgenetic strains have been derived after screening. Resistance analysis on these strains reveals that the strains overexpressing AhNAC2 gene have higher level of drought resistance than the wild strains. Besides, in normal condition, some downstream genes containing DRE/CRT element expressed more in the transgenetic strains than the wild strains. The above results indicate that AhNAC2 may induce the expression of abiotic stress related COR/RD genes. Besides, water loss analysis showed that the transgenetic strains were more resistant to drought. These results indicate a role of AhNAC2 in drought stress regulation. In conclusion, we have isolated two NAC transcription factors, AhNAC1 and AhNAC2, and verified their physiological functions. These results will not only help us to understand the mechanism of peanut resistant to drought, but also provide the gene for genetic improvement of peanut drought resistance.