Cloning And Functional Analysis Of TaFKBP62c-2B Gene In Wheat (Triticum Aestivum L.)

Wheat is one of the most important crops in the world, Its planting area and production is the highest in food crops. However, high temperature and drought occurred frequently, especially hot drought stress seriously affect the production of wheat in the early stage of the grouting. Cultivating heat and drought resistance wheat varieties is the most economic and efficient way. But, the heat and drought resistance belong to quantitative traits, wheat has large genome and complex mechanism, it is difficult to increase heat and drought resistance of wheat. Molecular breeding is the premise of the research. FK506-binding proteins(FKBPs) are well known as both the receptor for the immunosuppressant drug FK506 and the prolyl isomerase(PPIase)enzyme. FKBPs are widely and constitutively expressed, and highly conserved during evolution. Current researches about plant FKBP are mainly concentrated on a few plants such as Arabidopsis(Arabidopsis thaliana), rice(Oryza sativa L) and maize(Zea mays). Cloning and function analysis of wheat(Triticum aestivum L) FKBP genes were rarely reported. Research about wheat FKBP is still at the initial stage, thus, there is still a lot of work required to do about cloning and function analysis of wheat FKBP genes. In this study, the wheat TaFKBP62c-2B gene was cloned, expression pattern analysis were widely carried out under abiotic stresses, subcellular localization of TaFKBP62c-2B was detected, and its function was analyzed through the transformation of model plant Arabidopsis thaliana. The main results of this study are as follows:1. TaFKBP62c-2B wheat FKBP gene was cloned from wheat cDNA. The total length of the TaFKBP62c-2B gene was 1926 bp and the protein contained 642 amino acids. Bioinformatics analysis showed that TaFKBP62c-2B has three FKBP_C conserved domains( FK506 bindingprotein_C conserved domain), three TPR(tetratricopeptide repeat) and a transmembrance region at carbon terminal. Ta FKBP62c-2B belonged to multiple domain. Phylogenetic analysis showed that the homology value of FKBP62 c and other species reached 86%-96%。2. Expression patterns under different stresses of TaFKBP62c-2B gene was investigated using real-time quantitative PCR(qRT-PCR) assay. It was found that TaFKBP62c-2B gene was induced by high temperature and drought. TaFKBP62c-2B gene was induced by high temperature: TaFKBP62c-2B gene was induced after exposed to high temperature, and the expression value reached maximum at 2 h and then gradually declined; TaFKBP62c-2B gene was induced by drought: TaFKBP62c-2B gene was induced after exposed to drought stress, and the expression value reached maximum at 2 h, then expression gradually declined to minimum at 4 h and rised again.3. Tissues expression patterns of TaFKBP62c-2B gene was investigated using real-time quantitative PCR(qRT-PCR) assay. It showed that TaFKBP62C-2B expressed in root, stem, leaf, spike, spikelet, lemma and palea. Stem accumulated higher TaFKBP62C-2B transcription compared with the spike, spikelet, lemma and palea, while root and leaf had least TaFKBP62C-2B.4. The subcellular localization vector TaFKBP62c-2B::16318GFP was built, and then transferred into wheat mesophyll protoplast mediated by PEG. Using laser confocal microscope was used to detect subcellular localization of the target protein and 16318 GFP. As control, 16318 GFP was fluorescent in cytoplasm, nucleus, TaFKBP62c-2B was located in the cytoplasm.5. Transgenic Arabidopsis was obtained using transformation mediated by soil agrobacterium(Agrobacterium) containing plant expression vector pCAMBIA1302:: TaFKBP62c-2B. T3 generations of transgenic Arabidopsis plants or seeds were treated under drought stress. It was preliminary showed that the ability resisting to drought stress was improved in transgenic Arabidopsis due to overexpression of TaFKBP62c-2B gene. This work will provide the theoretical basis for transgenic breeding using TaFKBP62c-2B gene.