Identification Of Two NAC Genes And Their Expression And Function In Brassica Napus

Brassica napus belongs to Brassicaceae family, which is an important oilseed plant. Brassica napus is one of large amount agricultural products for our country economical trade. Therefore investigate the genes expression and molecular mechanism which are involved in response to abiotic stresses in Brassica napus, will make significantly for increasing Brassica napus produce. NAC proteins have been considered as one of the novel classes of plant-specific transcription factors. It has highly conserved DNA-binding domain in N-terminal, and highly variable C- terminal domain which contains the transcriptional activation domain, the N-terminal which can divide into A, B, C, D, E five subdomains, subdomains A, C, and D were tightly conserved, On the other hand, the diversity of the sequences in subdomains B and E. NAC transcription factors are not only involved in regulating plant development, but they were also response to biotic and abiotic stresses. In this study, two novel NAC genes were cloned in Brassica napus, consequently designated as BnNAC2, BnNAC5. Our research has focused on the expression pattern and function of the two BnNAC genes, the main results are as follows:1. Expression of BnNAC2 and BnNAC5 genes are upregulated by NaCl, mannitol and ABAIn order to study the NAC genes in Brassica napus, we obtain six full-length cDNA sequences encoding putative NAC-like proteins in B. napus by bioinformatics analysis, and designated as BnNAC1-6. First of all, the expression profiles of the six genes under salinity, drought and ABA were analyzed by real-time quantitative RT-PCR. The results demonstrated that BnNAC2 and BnNAC5 were significantly induced by salinity, drought and ABA, suggesting that the BnNAC1 and BnNAC5 genes may be involved in B.napus response to abiotic stresses, it was also indicated that a significant cross-talk may occur between drought and high-salinity stress signaling and between drought and ABA responses.2. Isolation and characterization of two BnNAC genes.We cloned the two NAC genes cDNA fragment of BnNAC2 and BnNAC5 from Brassica napus. Two genomic DNA of BnNAC2/5 were also acquired by PCR method in the Brassica napus genomic DNA.We analyzed the structure of two BnNACs and found that the two genes contain three extrons and two introns. An alignment of the predicted amino acid sequence of BnNACs with the cloned NAC-like genes from Arabidopsis was conducted, the results showed that higher sequence similarities are found in the N-terminus, which contained several distinguishable blocks of heterogeneous amino acids or gaps and are divided into five subdomains, the C terminus of BnNAC proteins are highly variable.3. Expression pattern of BnNAC genes in Brassica napus tissuesRNA was extracted from Brassica napus tissues and reversed transcription into cDNA. We analyzed the expression patterns of BnNAC2 and BnNAC5 though realtime RT-PCR. The results showed that the two genes expression in each tissues. BnNAC2 mRNAs were accumulated at relatively high levels in flowers. By contrast, BnNAC5 transcripts were accumulated at the highest levels in stems. We also cloned the promoter of BnNAC'2 gene, the BnNAC2promoter::GUS fusion expression vector was constructed and introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation. Expression of the GUS gene under the control of BnNAC2 promoter in transgenic Arabidopsis plants was examined by histochemical assay. The BnNAC2p:GUS expression patterns are consistent with RT-PCR results. These results suggested that two NAC genes probably play different functions during the processes of B.napus development.4. Transcriptional activity of BnNAC2 and BnNAC5We cloned the cDNA fragments containing the C-terminal domains of the two BnNAC TFs into pGBKT7 which contains the GAL4-binding domain, and transform the fusion plasmids into AH 109 yeast strain to analyed their transcription activation activity. These results suggest that the BnNAC2 protein functions as a transcriptional activator, while the BnNAC5 may be a transcriptional repressor.5. Overexpression of BnNAC2/5 in yeast enhances cell sensitivity to salt and drought stressTo investigate whether the BnNAC2 and BnNAC5 genes play roles in response to high-salinity and drought stresses, we cloned the coding sequence of two genes, constructed it into pREP5N vector, transformed into yeast cell. The transformed yeast cell lines treated with different conditions to evaluate cell growth status, the results showed that their growth was more sensitive to the stresses, compared to the control lines. Statistical analysis further confirmed the above results. Meanwhile, we also analyse the cell morphology of BnNAC2/5 overexpression in yeast, the results demonstrated that overexpression of the BnNAC2 gene stimulates the longitudinal growth of the host cells, and we also analysis cell division between the transformed lines and controls by DAPI stanining, statistical dates demonstrated that there were no significant difference.