The Molecular Characterization Of Wheat NF-Y Family Member TaNF-YB3;1and Its Roles In Mediating Plant Tolerance To Stresses Of Drought And Salt

The responses and tolerances of plants to environmental stresses are complishedthrough fine regulation of quantity of genes at transcriptional level via distincttranscription factors. Nuclear factor Y (NF-Y) is a type of typical transcription factorsin eukaryotes and plays an important role on regulating the transcription ofdownstream genes by interacting with a conserved motif (CCAAT) in gene promoters.In the current study, an expressed sequence tag (EST) of TaNF-YB3;1, a transcriptionfactor gene classified into NF-Y family, was used to perform further analysis. Theopen reading frame (ORF) of TaNF-YB3;1was cloned method and its molecularcharacterization and expression patterns were studied. Using the Agrobacteriumtumefactions-mediated genetic transformation approach, the transgenic tobacco plantswith overexpression of TaNF-YB3;1were generated on which the effects ofNF-YB3;1on regulation of plant tolerances to stresses of drought and salt wereinvestigated. The main results are as follows.1. Based on sequencing the clones in a wheat root subtractive cDNA library thatenriched drought tolerance-related genes, one expressed sequence tag (EST) thatshared high similarity to a wheat transcription factor NF-YB3was identified. Blastsearch analysis against the GenBank revealed the full length cDNA sequence of thisEST. The gene was denoted as TaNF-YB3;1herein. The open reading frame ofTaNF-YB3;1, was amplified based on reverse-transcription PCR in droughtstressed-wheat roots.2. The full-length cDNA of TaNF-YB3;1is790bp, containing an open readingframe of624bp and encoding a polypeptide of208-aa. TaNF-YB3;1has a molecularweight of18.05kD and an isoelectric point (pI) of7.14, containing the conserveddomian shared by NF-YB proteins, including the short ring that separates the3/4ofalpha helix and alpha helix.3. Based on homologous search analysis in GenBank, a subset of homologousgenes that share high similarities to TaNF-YB3;1were identified and subjected tofurther phylogenetic analysis. The results reveal that TaNF-YB3;1is homologous with various NF-YB3genes that are derived from rice (O. sativa), maize (Z. mays), barley(H. vulgare), phyllostachys (P. edulish), selaginella (P. Beauv), sitka spruce (P.sitchensis), soybean (G. Max), sorghum (S. Bicolor), and Garlic (A. sativum). Theseresults suggest that TaNF-YB3;1and above closely homologous gene are possiblyevolved from a same or close progenitor.4. Using wheat cultivar Shixin828as the materials, the expression patterns ofNF-YB3;1in roots were studied. Compared with that in normal growth condition, theexpression levels of NF-YB3;1were up-regulated under stresses of drought and salt.These results implicate that NF-YB3;1is possibly involved in tolerances of stresses ofdrought and salt through its responses to above abiotic stresses.5. Adopting DNA recombinant technology, a binary expression cassette tointegrate the open reading frame of NF-YB3;1into vector pCAMBIA3301wasconstructed. The binary cassette was then subjected to transform the Agrobacteriumtumefaciens strain EHA105via heat shock method. Furthermore,the transgenic tobacco plants with overexpression of TaNF-YB3;1was generatedthrough Agrobacterium tumefaciens-based genetic transformation method.6. The plants of wild type (Control) and typical transgenic tobacco plants withoverexpression of TaNF-YB3;1were subjected to stresses of drought and salt, onwhich to investigate the function of TaNF-YB3;1on regulating plant tolerances toabove stresses. The results indicate that the transgenic plants exhibited strongertolerances to drought and salt than the wild type plants, showing much larger plantfeatures and much more accumulation of plant dry mass. Thus, it is confirmed thatTaNF-YB3;1acts as an critical regulator in plant tolerances to stresses of drought andsalt.7. After stresses of drought and salt, a subset of abiotic stress-relatedphysiological parameters were varied in the transgenic plants in contrast to those ofthe wild type plants. Compared with those in the wild type plant, the transgenic plantswith everexpression of TaNF-YB3;1behaved higher contents of chlorophyll andsoluble protein as well as higher activities of superoxide dismutase (SOD) andperoxidase (POD). These results suggest that the improved tolerances of drought andsalt in the TaNF-YB3;1-ovexpressing plants are partly associated with the improvedphysiological parameters such as those of contents of chlorophyll and soluble proteinas well as those of activities of SOD and POD. Our current study demonstrate thatTaNF-YB3;1can be used as one of elite gene resources in evaluation and screening ofstresses of drought and salt among wheat genetic materials. It is also useful gene ingenerating novel germplasms of wheat as well as possibly other crops with strongertolerances to drought and salt.