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.
[1]杨绚, 汤绪, 陈葆德, 等.气候变暖背景下高温胁迫对中国小麦产量的影响[J].地理科学进展, 2013, 32(12):1771-1779
[2]于彦丽, 李艳娇, 庞凯元, 等.植物基因家族的结构及生物学功能[J].遗传, 2014, 36(6):536-546
[3]Ahn J C, Kim D W, You Y N, et a1.Classification of rice(Oryza sativa L. Japonica nipponbare) immunophilins(FKBPs, CYPs) and expression patterns under water stress[J]. BMC plant biology, 2010, 10(1): 253.
[4]Aviezer-Hagai K, SkoVorodnikova J, Galigniana M, et a.Arabidopsis immunophilins ROF1(AtFKBP62)and ROF2(AtFKBP65) exhibit tissue specificity,are heat-stress induced,and bind HSP90[J].Plant molecular biology, 2007, 63(2):237-255
[5]Bierer B E, Patricia K S, Thomas J W, et al.Probing immunosuppressant action with a non-natural immunophilin ligand[J]. Science, 1990, 250: 556-559.
[6]DeCenzo M T, Park S T, Jarrett B P, et al.FK506-binding protein mutational analysis: defining the active-site residue contributions to catalysis and the stability of ligand complexes[J].Protein engineering, 1996, 9(2):173-180
[7]Ellis R J.Molecular chaperones: the plant connection[J]. Science, 1990, 250: 954-959.
[8]Faure J D, Gingerich D, Howell S H.An Arabidopsis immunophilin, AtFKBP12, binds to AtFIP37 (FKBP interacting protein) in an interaction that is disrupted by FK506[J]. The Plant Journal, 1998, 15: 783-789.
[9]Fruman D A, Burakoff S J, Bierer B E.Immunophilins in protein folding and immunosuppression[J].The FASEB journal, 1994, 8(6):391-400
[10]Fischer G, Wittmann-Liebold B, Lang K, et al.Cyclophilin and peptidyl-prolyl cis–trans isomerase are probably identical proteins. Nature, 1989, 337: 476-478.
[11]Freskgard P O, Bergenhem N, Jonsson B H, et al.Isomerase and chaperone activity of prolyl isomerase in the folding of carbonic anhydrase. Science, 1992, 258: 466-468.
[12]Geisler M, Bailly A.Tete-a-tete: the function of FKBPs in plant development [J]. Trends in plant science, 2007, 12: 465-473.
[13]Geisler M, Girin M, Brandt S, et al.Arabidopsis immunophilin-like TWD1 functionally interacts with vacuolar ABC transporters[J].Molecular biology of the cell, 2004, 15(7):3393-3405
[14]Gollan P J, Bhave M.Genome-wide analysis of genes encoding FK506-binding proteins in rice[J]. Plant molecular biology, 2010, 72(1-2), 1-l6.
[15]Harding M W, Galat A, Uehling DE, et al.A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase[J]. Nature, 1989, 341: 758-760.
[16]He Z, Li L, Luan S.Immunophilins and parvulinsSuperfamily of peptidyl prolyl isomerases in Arabidopsis[J].Plant physiology, 2004, 134(4):1248-1267
[17]Kurek I, Aviezer K, Erel N, et al.The wheat peptidyl prolyl cis-trans-isomerase FKBP77 is heat induced and developmentally regulated[J]. Plant Physiol, 1999, 119: 693-704.
[18]Nigam N, Singh A, Sahi C, et al.Sumo-conjugating enzyme (Sce) and FK506-binding protein (FKBP) encoding rice (Oryza sativa L.) genes: genome-wide analysis, expression studies and evidence for their involvement in abiotic stress response[J]. Molecular Genetics and Genomics, 2008, 279: 371-383.
[19]Magiri EN, Farchi-Pisanty O, Avni A, et al.The expression of the large rice FK506 binding proteins(FKBPs) demonstrate tissue specificity and heat stress responsiveness[J].Plant science, 2006, 170(4):695-704
[20]Maas C, Werr W.Mechanism and optimized conditions for PEG mediated DNA transfection into plant protoplasts[J].Plant cell reports, 1989, 8(3):148-151
[21]Meiri D, Breiman A.Arabidopsis ROF1(FKBP62)modulates thermotolerance by interacting with HSP90[J].and afecting the accumulation of HsfA2-regulated sHSPs. The Plant Journal, 2009, 59(3):387-399
[22]Meiri D, Tazat K, Cohen-Peer R, et a.Involvement of Arabidopsis ROF2(FKBP65) in thermotolerance[J].Plant molecular biology, 2010, 72(1-2):191-203
[23]Nigam N, Singh A, Sahi C, et a.SUMO-conjugating enzyme (Sce) and FK506-binding protein(FKBP)encoding rice(Oryza sativa L) genes: genome-wide analysis,expression studies and evidence for their involvement in abiotic stress response[J].Molecular Genetics and Genomics, 2008, 279(4):371-383
[24]Perez-Perez J, Ponce M, Micol J.Mutations in the ULTRACURVATA2 gene of Arabidopsis thaliana, which encodes a FKBP-like protein, cause dwarfism, leaf epistasy and helical rotation of several organs[J]. International Journal of Developmental Biology, 2001, 45: S49-S50.
[25]Schreiber S L.Chemistry and biology of the immunophilins and their immunosuppressive ligands[J]. Science, 1991, 251: 283-287.
[26]Siekierka J J, Staruch M J, Hung S H, et al.FK-506, a potent novel immunosuppressive agent, binds to a cytosolic protein which is distinct from the cyclosporin A-binding protein cyclophilin[J]. J Immunol, 1989, 143: 1580-1583.
[27]Takahashi N, Hayano T, Suzuki M.Peptidyl-prolyl cis-trans isomerase is the cyclosporin A-binding protein cyclophilin[J]. Nature, 1989, 337: 473-475.
[28]Vespa L, Vachon G, Berger F D R, et al.The immunophilin-interacting protein AtFIP37 from Arabidopsis is essential for plant development and is involved in trichome endoreduplication[J]. Plant Physiol, 2004, 134: 1283-1292.
[29]Wu G S, Otegui M S, Spalding E P.The ER-localized TWD1 immunophilin is necessary for localization of multidrug resistance-like proteins required for polar auxin transport in Arabidopsis roots[J].Plant Cell, 2010, 22(10):3295-3304
[30]Yoo S D, Cho Y H, Sheen J.Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[J]. Nature protocols, 2007, 2: 1565-1572.
[31]Yu Y, Li Y, Huang G, et al.PwHAP5, a CCAAT-binding transcription factor, interacts with PwFKBP12 and plays a role in pollen tube growth orientation in Picea wilsonii[J]. Journal of experimental botany, 2011, 62: 4805-4817.
[32]Yu Y L, Zhang H, Li W C, et a.Genome-wide analysis and environmental response profiling of the FK506-binding protein gene family in maize(Zea mays L)[J].Gene, 2012, 498(2):212-222
[33].
