| Plants are exposed to many types of abiotic stress and biotic stress during their life-cycle, such as drought, salinity, and high and low temperatures, which have adverse effects on growth and the productivity of crop plants. A variety of plant genes are induced by a single or multiple stresses, and their products are thought to function in stress tolerance and in the regulation of gene expression and signal transduction in stress responses. Transcription factors have critical roles in the regulation of gene expression. Various transcription factors and cis-acting elements in the stress-responsive promoters function in plant adaptation to environmental stress. AP2/ERF is a family of transcription factors containing the highly conserved ethylene-responsive-element-binding AP2 DNA-binding domain. The AP2/ERF family genes are involved in response to drought, high salt content, temperature change, disease resistance, and the flowering control pathway, and have been analyzed by a combination of genetic and molecular approaches.Analysis of plant genomes can also facilitate the study of genome and gene evolution. Until relatively recently, gene functional dissection of plant has depended largely on molecular characterization of individual genes from various species. Companied with complete genome sequences, technologies of bioinformatic analysis have advanced so that the information stored in the various genomes of plants can be explored to search information that regulate plant growth and development. Brassica rapa, B. napus and Arabidopsis thaliana are members of the crucifereae and the protein coding sequences are conserved among the three species. In this study, we cloned and analyzed an important plant transcription factor family, AP2/ERF from Chinese cabbage, oilseed and compared them with the model plant species, Arabidopsis. Since AP2/ERF gene family are involved in regulating stress responses and flowering, and relatively few genes of this gene family from Chinese cabbage and oilseed have been characterized in detail, these bioinformatic analyses will help scientists to make use of information from other species to study those plants stress resistance and flowering and fruit development, such as orthorlogous relationship of a particular stress response gene.1. Starting from a large database of B. rapa expressed sequence tags (ESTs),62 putative AP2/ERF family genes were identified by in silico cloning using the amino acid sequence of A. thaliana as a probe. Based on the number of AP2/ERF domains and functions of the genes, the AP2/ERF transcription factors from B. rapa were classified into four subfamilies as ERF, DREB, RAV and AP2. Nearly all the genes were similar to those defined earlier as AP2/ERF from A. thaliana. The following properties and attributes were predicted and analyzed:cDNA and deduced amino acid sequence, composition, physical and chemical characterization, phylogenetic tree, conserved domain sequences, and function domain. Using large-scale transcriptome analysis and available EST information as a source of expression data for digital expression profiling, differentially expressed genes were identified among diverse plant tissues. Roots contained the largest number of transcripts of the AP2/ERF family genes, followed by leaves and seeds. Only a few of the 62 AP2/ERF family genes were expressed in all tissues:most were expressed only in some tissues but not in others. Some AP2/ERF family genes were constitutive and expressed in all the six kinds of tissues investigated namely seeds, siliques, buds, flowers, leaves, and roots. The maximum expression was that of BraERF-B2-5, and it was recorded from seed tissue.2. Three AP2/ERF family transcriptional regulators (BnaERFl, BnaERF2 and BnaERF3) were isolated from B. napus by in silico cloning method using the conserved domain amino acid sequence of A. thaliana ERF as probe. Then, BnaERFl, BnaERF2 and BnaERF3 genes were isolated from B. napus L. Huyoul5 by RT-PCR and PCR using cDNA and DNA as template.3. Two AP2/ERF family transcriptional regulators (BnaERFB3-1 and BnaERFB3-2) were isolated from B. napus by in silico cloning method using the conserved domain amino acid sequence of A. thaliana AP2/ERF-B3 subfamily as probe. Based on the sequences of BnaERFB3-1 and BnaERFB3-2, we isolated the BnaERFB3-1-Hy15 gene and BnaERFB3-2-Hy15 gene from winter and spring type B. napus L. cv Huyoul5 by RT-PCR and PCR using cDNA and DNA as template. No intron localized on the two genes from Huyou15, respectively. BnaERFB3-1-Hy15 and BnaERFB3-2-Hy15 were hydrophilic protein. The two proteins and AtERF5 have similar three-dimension structure. The disordered residues of protein BnaERFB3-1-Hy15 and BnaERFB3-2-Hy15 were higher than that of AtERF5. BnaERFB3-1 was mainly expression in seed, while BnaERFB3-2 was mainly expression in root. Moreover, those genes were successfully constructed into the recombinant plasmids of plant expression vector and yeast expression vector.4. One AP2/ERF family transcriptional regulators (Named BnaERFB4-1) was isolated from B. napus by in silico cloning method using the conserved domain amino acid sequence of A. thaliana ERF-B4 subfamily as probe. BnaERFB4-1 was hydrophilic protein. The protein and RAP2.6L have similar three-dimension structure. The disordered residues of protein BnaERFB4-1 was higher than that of RAP2.6L. We isolated the BnaERFB4-1 gene from winter and spring type B. napus L. cv Huyou15 by RT-PCR and PCR using cDNA and DNA as template. DNA sequencing and analyzing indicated that there only three amino acid residues difference between BnaERFB4-1-Hy15 and BnaERFB4-1. Moreover, the gene was ligated into expression vectors, respectively. It was successful to construct the recombinant plasmids of plant expression vector and yeast expression vector.5. Two CBF family transcriptional regulators were isolated from B. napus by in silico cloning method using the conserved domain amino acid sequence of AtCBF1 as probe. Based on the sequences of BnaCBF-1 and BnaCBF-2 from Bna.2193 and Bna.2260, the BnaCBF-1-Hy15 and BnaCBF-2-Hy15 genes were isolated from B. napus L. cv Huyou15 by RT-PCR and PCR using cDNA and DNA as template. No intron localized on the two genes from Huyou15, respectively. BnaCBF-1-Hy15 and BnaCBF-2-Hy15 were part of the DREB-A1 subfamily, and were hydrophilic protein. The two proteins and AtCBF1 have similar three-dimension structure. |
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