| Transcription factors(TFs) play an important role in regulating plant responses to biotic and abiotic stresses, and in mediating plant growth and development, and signal transduction. Nuclear factor Y(NF-Y) is a type of transcription factors which regulate the expression of downstream target genes by binding to the CCAAT-box in promoters of target eukaryotic genes. In this study, based on the expressed sequence tag(EST) of NF-Y type transcription factor Ta NF-YB2;1 that identified in the wheat roots subtractive suppression c DNA library pre-constructed in the laboratory, Ta NF-YB2;1 was cloned. Molecular characterization, expression patterns under drought and salt stresses, and biological functions of Ta NF-YB2;1 mediating plant responses to drought, salt and other abiotic stresses were systematically studied. In addition, the binary expression vectors fused the sense and antisense sequence of four C2H2-type transcription factor genes, Ta ZFP1~Ta ZFP4, were constructed in this study. These binary expression vectors were then subjected to transform tobacco via an Agrobacterium-mediated genetic transformation approach. The work thus has provided the basis for further understanding the biological functions of C2H2-type TF members in wheat. The main results are as follows.1. One expressed sequence tag(EST) sharing high similarity to transcription factor gene NF-YB2-like in Brachypodium distachyon was identified in a wheat root subtractive suppression hybridization c DNA library that enriches drought stress. The c DNA sequence corresponding to this EST was obtained based on homologous search in NCBI and named Ta NF-YB2;1. The full-length c DNA of Ta NF-YB2;1 is 958 bp, encoding a 163 amino acid-polypeptide. The molecular weight of Ta NF-YB2;1 is 17.74 k D with isoelectric point(p I) of 6.13. The protein encoded by this gene contains four conserved α-helix motifs, α1, α2, α3 and αC, which are shared by NF-YB2 family members in plant species.2. Based on homologous search in NCBI, 23 homologous genes of Ta NF-YB2;1 in various plant specie were identified and subjected to further phylogenetic analysis using DNAStar software. Ta NF-YB2;1 was shown to share high similarities with NFYB-B2 in Triticum aestivum, NF-YB2-like in Brachypodium distachyon, Os HAP3 A in Oryza sativa at nucleic acid level, with the identities of 97.1%, 80.7% and 70.7% respectively. These results suggest that Ta NF-YB2;1 and above homologous genes possibly share similar evolutionary relationship or derive from close ancestors.3. The expression patterns of Ta NF-YB2;1 in wheat roots under treatments of drought and salt were analyzed. After drought or salt stresses, the transcripts of Ta NF-YB2;1 drastically increased compared with those in normal condition, suggesting that Ta NF-YB2;1 may play an important role in mediating plant reponse to above stresses.4. Using the Agrobacterium-mediated genetic transformation approach, the transgenic tobacco plants overexpressing Ta NF-YB2;1 were generated. Using typical transgenic tobacco lines of Ta NF-YB2;1 and wild type tobacco as materials, the phenotype fetures and dry mass of the plants under drought and salt stresses were studied. The plants overexpressing Ta NF-YB2;1 exhibited significantly improved growth features and dry mass compared with wild type plants under above treatments, which implied genetic transformation of Ta NF-YB2;1 can significantly improved tobacco plant to resist drought and salinity stresses.5. Using transgenic lines with sense(Sen-2) and antisense(Ant-1) expressions of Ta NF-YB2;1 and wild type(WT) as materials, the expression patterns of genes encoding cellular antioxidant enzymes, phosphate transporters and nitrate transporters were investigated and the characteristics of related physiological parameters under drought and salinity were analyzed. The results showed that the transcripts of part SOD and CAT related genes in Sen-2 were drastically increased in comparison with those in WT, which were in consistent with the higher activities of SOD and CAT and higher content of soluble protein in the transgenic plants. The expressions of most POD genes were shown to be decreased in Sen-2 plants were also in agreement with the lower POD activities in the transgenic plants than WT under the above stresses. The behabiors of gene expression and antioxidant enzymatic activities in the Ant-1 plants were generally opposite to those in the Sen-2 plants. The transcripts of few NTPT and NTNRT genes were shown varied amount among the plants of Sen-2, Ant-1 and wild type. These results indicated that Ta NF-YB2;1 is a critical regulator in mediating plant tolerance to drought and high salinity through modifying expressions of distinct genes encoding cellular antioxidant enzymes, phosphate transporters and nitrate transporters, on which to regulate the physiological and biochemical processes associated with plant tolerance to aforementioned stresses. Therefore, Ta NF-YB2;1 mediate plants to tolerate the stresses by integrated the complicated regulatory networks through its transcriptionally regulate the downstream defensive-related genes.6. The accumulation of H2O2 and O2-· in leaves of the Sen-2, Ant-1 and WT plants after drought and salt treatments were analyzed by DAB and NBT histochemical staining methods. Less amount of above reactive oxygen species(ROS) was detected in the Sen-2 leaves than in the WT plants, indicating that overexpression of Ta NF-YB2;1 can enhance the ability of plants to scavenge the ROS initiated by stresses of drought and high salinity. In contrast, darker color of Ant-1 leaves after stained than leaves of Sen-2 and WT suggested that downregulated expression of the Ta NF-YB2;1 homolog can reduce the ability to remove ROS under the drought and salt stress conditions.7. Based on sequencing the clones in a wheat root subtractive suppression c DNA library that enriched low-Pi responsive genes under various time points, four expressed sequence tags(ESTs) classified into the C2H2-type TF family were identified. The c DNA sequences corresponding to these ESTs were obtained based on homologous search and RT-PCR. They were named as Ta ZFP1~Ta ZFP4 in this study(Accession number: AK331121, AK333490, AK334138 and AK333024). The full-length c DNA of Ta ZFP1, 2, 3 and 4 were 708 bp, 1507 bp, 1612 bp and 940 bp, respectively, and they separately encode the polypeptides with amino acids of 124-aa, 400-aa, 383-aa and 119-aa. Prediction analysis revealed that the molecular weight of Ta ZFP1, 2, 3, and 4 were 13.96 k D, 45.01 k D, 44.09 k D and 12.42 k D, respectively, with the isoelectric point(p I) of 9.3, 5.27, 7.54 and 9.97.8. Homologous search analyses were performed to obtain the homologous genes of Ta ZFP1~Ta ZFP4 in various plant species. The phylogenetic tree covering Ta ZFP1~Ta ZFP4 and their homologous genes were generated. The results showed that Ta ZFP1~Ta ZFP4 were highly homologous with those genes derived from Hordeum vulgare, Brachypodium distachyon and Zea mays. Thus, the ZFP genes in wheat are suggested to be sharing closely genetic relationship with their himologous partners in other plant species.9. Expression patterns of Ta ZFP1~Ta ZFP4 were investigated under various treatments such as low-Pi, low-N, drought and high salinity. It was shown that a set of them were significantly regulated by part or all of the above treatments. Using RT-PCR and DNA recombinant technology, the binary expression plasmids fusing the sense and antisense sequences of Ta ZFP1~Ta ZFP4 were separately constructed. Transgenic tobacco plants fused above sense and antisense sequences of TF genes were generated via Agrobacterium tumefaciens-based genetic transformation method. These plants have provided a solid basis for further defining the biological functions of Ta ZFP1~Ta ZFP4 in mediating plants tolerance to aforementioned abiotic stresses in future. |
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