Clone, Expression And Transformation Of The NAC Transcription Factor Gene In Bothriochloa Ischaemum

NAC (NAM, ATAF, and CUC) is found as a class of plant-specific transcription factor family for more than10years, involving in plant growth and development, aging, hormonal response, secondary metabolism and responses to stress. A highly conservative NAC domain in N-Terminal is the main structural characteristics of all NAC transcription factors contaaining about150amino acid residues. Bothriochloa ischaemum is a kind of fodder pasture. In order to understand molecular mechanism of B. ischaemum. In this paper, one NAC transcription factor gene was cloned by RT-PCR and RACE methods from B. ischaemum, and the sequence characters, expression patterns and functions were deeply analyzed to understand clearly molecular mechanism of plant response to various abiotic stresses. The main results are follows:1. Using RT-PCR and RACE technology, a cDNA of NAC gene was cloned from B. ischaemum, named BiNAC, and the Genbank accession number is KF030136. The gene’s code area has1125bp in length which encode374Amino Acids.2. Analyze sequense show:BiNAC gene has an introne and two exon, BiNAC protein has the typical characteristics domains of NAC transcription factor. The deduced amino acid sequences of BiNAC showed significant sequence similarity with those of other known NACs. Phylogenetic tree analysis indicated that BiNAC gene belongs to the ATAF subfamily, and the BiNAC gene of B. ischaemum was closely related to Sorghum bicolor with94%homology.3. Analyze specificity expression of BiNAC gene in different organization by Real-time PCR. Result show that it has some discrepancies in three organizations. It was expressed strongly in stem, and faintly in roots.4. Analyzed to understand clearly expression patterns of B. ischaemum response to various abiotic stresses by real time PCR. Consequenses show:expression of BiNAC gene not only be induced by NaCl, but also induced by exogenous hormone ABA. No significant changes in the BiNAC expression were observed under PEG, low temperature and Dark. Thereafter, BiNAC gene may be involved in the defense responses against adversity of B. ischaemum. 5. We constructed the fusion protein expression vector pCAMBIA1302-BiNAC-GFP, Analyzed result show:BiNAC protein was located in the cell nucleus. Fusion protein pCAMBIA1302-BiNAC-GFP was mainly distributed in the cell nucleus. But pCAMBIA1302were distributed in the whole cell. It is demonstrated that BiNAC protein has the trait of nuclear localization.6. We constructed the fusion protein expression vector pCAMBIA1302-BiNAC-GFP and pBI121-BiNA4C-GUS, We transformed BiNAC gene into Arabidopsis with Agrobacterium tumfaciens containing an expression vector by Agrobacterium infestation. Detect T1generation of transgenic Arabidopsis by PCR and RT-PCR. The results showed:BiNAC gene has been transformed into Arabidopsis and expressed in it.