| Plants have a series of mechanisms to responsive to environment, and annexin is one of them. It plays an important role in stress response during plant growth and development. In the research, a novel tartary buckwheat annexin gene FtANX1 and its promoter were obtained. And we conducted a survey of FtANX1 structure, phylogenetic analysis, the subcellular distribution, and gene expression patterns. Our results indicated that FtANX1 might play an important role in various stresses, and would provide a basis for further utilize in plants. The main results are as follows:1. Based on RACE cloning, a tartary buckwheat annexin gene was obtained, named FtANX1. The full-length DNA sequence of it was 1650 bp. Bioinformatic analysis suggested that it contains five exons and four introns. The CDS was 942 bp, encoded 313 amino acid residues. The molecular mass is 36.06 KDa, and the isoelectric point is 5.94. This protein harbors four conserved annexin domains, and all a-helix. A typical type Ⅱ Ca2+binding site (G-X-G-T-[38]-E) was found. Multiple alignments between this annexin and other plant annexins showed a sequence similarity ranging from 49 to 60%. Post-translational modification site prediction showed that 10 potential phosphorylation sites, an N-myristoyl site, and a unique cell attachment site were found. Predicted by Swiss-Model, the three-dimensional structure of FtANX1 was similar to AnnCaP32 with the most (50.16%). According to cluster analysis, the plant annexin is divided into four groups, and tartary buckwheat annexin FtANX1 belongs to group II, with cotton AnnGh5, strawberry Annfaf, and alfalfa AnnMt1.2. Using the genome walking technique, the promoter sequence of FtANX1 gene were obtained. The predicted transcription start site was at 54 bp upstream from the start codon, the TATA-box element was at 31 bp upstream from the transcription start site. Cis-acting elements analyses were conducted by PLACE database, indicating that the promoter contains lot of elements which involved in biotic and abiotic stress.3. The expression pattern of FtANX1 gene in flowering tartary buckwheat tissues was conducted by qRT-PCR analysis. FtANX1 gene transcripts were detected in all tissues with the highest expression in immature seeds, relatively less abundant in roots, stems, flowers and mature seeds, and the least in leaves. These results indicated that tartary buckwheat annexin gene may be related to fruit development.4. The expression patterns of FtANXl gene in different stresses were performed by qRT-PCR. It showed that:comparing to the control, the expression of FtANX1 was up-regulated under JA and UV-B treatment, to a maximum of 9.79±0.89 and 10.71±0.40 at 24 h, respectively; after NaCl stress, the transcript levels were up-regulated, higher at 12 h; cold and drought resulted in mRNA accumulation; exposure to copper and lead caused a strong upregulation; zinc caused an increase 7 d after treatment, while a significant decrease 14 d after treatment; for iron and cadmium, the expression level was down-regulated 7 d, while induced 14 d; and nickel or manganese treatment caused a significant decrease in the gene expression of FtANX1. Our results indicate that FtANXl gene plays an important role in response to environmental stresses.5. Through the GFP subcellular localization system, a recombinant plasmid p163-GFF-FtANX1 was constructed and transformed into tobacco. The results showed that FtANX1 protein located in the cell membrane and might associated with cell differentiation, extracellular secretion, in response to environmental stress and so on. |
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