Cloning And Abiotic Stresses Functional Analysis Of Narcissus Tazetta ‘Yunxiang' NAC Transcription Factor

NAC transcription factors?NAM,ATAF1/2,CUC2?are widely distributed in plants,which have been identified to play many important roles in plant development regulation,as well as in both abiotic and biotic stress adaptation,and have been used in genetic engineering to improve plant stress tolerance.Narcissus tazetta var.Chinensis is one of the main ornamental flowers in winter,and its yield and quality are largely affected by external environmental factors.At present,little is known about the regulation of the Narcissus tazetta NAC transcription factor under stress,so it is important to cloning and identify good abiotic stress-related NAC transcription factors.In the current paper,we used the new species Narcissus tazetta‘Yunxiang'as the experimental material.and two NtNAC?NtNAC1 and NtNAC2?genes were cloned by RT-PCR.NtNAC gene was further characterized by analyzing its tempo-spacial expression patterns in Narcissus tazetta‘Yunxiang'plants,as well as changes in its expression in leaves and roots under different stresses and stress-related hormones,and by analyzing the gene over-expressing transgenic tobacco plants.The main results are as follows:A gene,designated as NtNAC1,was cloned by RT-PCR from roots of Narcissus tazetta‘Yunxiang'.The results showed that the clonedfull-length cDNA was 1 083bp long,containing an ORF of 918 bp long which would putatively encode a peptide of 306 amino acid residues.The deduced protein possessed a highly conserved DNA binding domain,NAM,at its N-terminal,and was grouped,together with its most closely related proteins,MusaSNAC1 and TaNAC47,into the SNAC1 subgroup on the phylogenetic tree.Quantitative real-time PCR Results showed that the expression level of the NtNAC1 in petal and corona first increased and then decreased as the floral organs developed,and reached a peak in the early flowering period.NtNAC1 was abundantly expressed in roots and leaves,and was induced by treatments of ABA,MeJA,SA,H₂O₂,NaCl and PEG,indicating that it may be involved in the morphogenesis of floral organs in addition to its principal role in the responses of plants to abiotic stresses.Nine transgenic tobacco lines carrying the NtNAC1 gene were obtained by agrobacterium-mediated transformation and three of them were over-expressing lines.The NtNAC1 over-expressing lines showed better developed root systems and higher survival rates under treatments of high concentrations of salt and mannitol,as compared with the non-transgenic plants.In addition,overexpression of transgenic lines in the transgenic lines P5CS,APX up-regulated expression,SOD,POD activity and MDA content decreased,reducing cell membrane damage caused by stress.Another NAC gene was cloned by RT-PCR and named NtNAC2,which contains 936 bp open reading frame encoding 312 amino acids.Bioinformatics analysis showed that NtNAC2 gene is a member of NAC family,which contains a typical NAM conserved domain located in the N-terminal region and a close evolutionary relationship grouped with monocotyledonous Asparagus officinalis,Crocus sativus,Dendrobium catenatum.The quantitative real-time PCR detection showed that the relative expression level of NtNAC2 gene in petal and corona increased gradually with the development of floral organs,and reached the peak in the senescence period.The expression profiles of NtNAC2 in root and leaf were analyzed,it was inducible under ABA,MeJA,SA,H₂O₂,50?,PEG and NaCl treatments,and the spatio-temporal expression difference of root and leaf was reflected.In order to further identify its function,we successfully constructed the plant over-expression vector and finally obtained 10 strains of transgenic tobacco.The over-expression of NtNAC2 in transgenic plants was detected by semi-quantitative RT-PCR.The roots of over-expression transgenic plants are 2 to 3 times that of wild type,the water loss rate of leaves is less than 30%and the survival rate is higher than 60%,and the transcriptional levels of stress-resistant related genes P5CS and APX increased,MDA has less accumulation,and SOD,POD have higher activity under the salt and drought stress.Our results showed that expressing NtNAC1 and NtNAC12 increases stress tolerance of plants and its potential in the improvement of abiotic stress resistance of Narcissus tazetta could be further exploited.