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| Cloning, Expression and Subcellular Localization of TaFKBP62c-2B Gene in Wheat (Triticum aestivum) |
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Abstract FK506-binding proteins (FKBPs) are known as the receptor for the immunosuppressant drug FK506. FKBPs are ubiquitously distributed in all living organisms and highly conserved during evolution. FKBPs have peptidyl-proly cis-trans isomerase (PPIase) activity and can be used as molecular chaperone to have a function in protein folding. Plant FKBPs take part in diversity of cellular processes including plant growth and abiotic stress responses. In this study, TaFKBP62c-2B was isolated from wheat (Triticum aestivum) by RT-PCR and the expression pattern of this gene was analyzed through qRT-PCR. The subcellular localization of TaFKBP62c-2B was also investigated by using the green fluorescent protein (GFP) method. The sequencing analysis showed that TaFKBP62c-2B gene consisted of 1 926 bp CDS, 37 bp 5' UTR and 176 bp 3' UTR, and encoded 642 amino acids (GenBank accession No: KU350629). The amino acid sequence analysis demonstrated that TaFKBP62c-2B had 3 conserved FK506 binding domains (FKBd), which were respectively named 62c-2BⅠ, 62c-2BⅡand 62c-2Ⅲ, and 3 tetratricopeptide repeats (TPR), so TaFKBP62c-2B belonged to multiple-domain FKBPs. The amino acid alignment analysis showed that 62c-2BⅠ was the most similar conserved domain with HsFKBP12a and should be the key domain for the PPIase function of TaFKBP62c. The 9 homologus genes from 9 different species of Aegilops tauschii, Triticum urartu, millet (Setaria italic), barley (Hordeum vulgare), Brachypodium distachyon, sorghum (Sorghum bicolor), maize (Zea mays), soybean (Glycine max), rice (Oryza sativa indica Group) of TaFKBP62c-2B were searched and found in NCBI database and the identity of amino acid between 9 homologous genes and TaFKBP62c-2B ranged of 86%~96%. The highest identity of amino acid was between EMS49100.1(T. urartu) and TaFKBP62c-2B, while the lowest was between soybean (XP_014634970.1) and TaFKBP62c-2B. Then phylogenetic tree of FKBP62c was constructed using these 9 genes and TaFKBP62c-2B. The expression pattern disclosed the expression level of TaFKBP62c-2B was increased immediately under heat stress, it came to the peaks at 2 h and droped gradully after that. TaFKBP62c-2B was responded to drought stress, the expression came to the peaks at 1 h and droped, it came to another peak at 8 h. Tissue expression pattern analysis showed that TaFKBP62c-2B expressed in multiple tissues. Stem accumulated higher TaFKBP62c-2B transcription compared with the spike, spikelet, lemma and palea, while root and leaf had the least TaFKBP62c-2B transcription among all the tissues. According to the prediction database, TaFKBP62c-2B was localized to endoplasmic reticulum (ER). The subcellular location were validated by means of fusing TaFKBP62c-2B with N-terminal GFP and then expressed in wheat protoplats using PEG transfection method. The green fluorescence were mainly observed in cytoplasm, and were not seen in chloroplasts. TaFKBP62c-2B should be localized to ER. In this study, the TaFKBP62c-2B gene was isolated, which the expression pattern and subcellular localization were detected. All above results pave the way to further function analysis of TaFKBP62c-2B.
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Received: 13 January 2016
Published: 20 May 2016
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