Abstract：DEAD-box RNA helicase (RH) ubiquitously exists in most organisms and participates, if not all, steps of cellular RNA metabolism. Generally, containing 9 highly conserved motifs is an important standard to identify a DEAD-box RNA helicase. In the present study, the CDS of a rice (Oryza sativa L. japonica. cv. Nipponbare) gene OsRH37, encoding a putative DEAD-box RNA helicase, was cloned. The amino acid sequence of OsRH37 was analyzed by bioinformatics method and it was indicated that the amino acid sequence identity between OsRH37 and 3 other Arabidopsis thaliana DEAD-box RNA helicases including AtRH17, 37 and 52 was＞70%, whereas the amino acid sequence identity between OsRH37 and DEAD-box RNA helicases from different animals was generally＜60%. The overexpression vector and RNAi vector of OsRH37 were constructed, and by using rice genetic transformation system the overexpression and RNAi transgenic rice plants of OsRH37 were obtained. Six lines (66.7%) among 9 lines of OsRH37 overexpressed transgenic rice and 13 lines (92.9%) among 14 lines of OsRH37 RNAi transgenic rice were identified to be positive, respectively. The relative expression level of OsRH37 among wild type, overexpression lines, and RNAi lines were detected by sqRT-PCR. The results indicated that compared with the wild type (WT), the expression level of OsRH37 in overexpression lines was obviously up-regulated while that in RNAi lines was significantly down-regulated. To observe the subcellular localization pattern of OsRH37, the stop codon TAG deleted CDS was constructed into the transient expression vector pEarleyGate102, harboring a cyan fluorescence protein (CFP), by Gateway technology and the leaves of Nicotiana benthamiana agroinfiltrated with CFP-OsRH37 were observed under confocal microscope at 48 h post infiltrated. The results of subcellular localization analysis suggested that OsRH37 fused with CFP was mostly localized at nucleus, karyosome and cytoplasmic granular structures. The bioinformatics analysis, obtained transgenic rice plants and subcellular localization analysis of OsRH37 can provide foundational insights for the important roles of OsRH37 in the processes of the rice growth, development and stress responses.
Aubourg, S., Kreis, M., and Lecharny, A. (1999). The DEAD box RNA helicase family in Arabidopsis thaliana. Nucleic acids research 27, 628-636. Blum, S., Schmid, S.R., Pause, A., Buser, P., Linder, P., Sonenberg, N., and Trachsel, H. (1992). ATP hydrolysis by initiation factor 4A is required for translation initiation in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America 89, 7664-7668. Boudet, N., Aubourg, S., Toffano-Nioche, C., Kreis, M., and Lecharny, A. (2001). Evolution of intron/exon structure of DEAD helicase family genes in Arabidopsis, Caenorhabditis, and Drosophila. Genome research 11, 2101-2114. Cordin, O., Banroques, J., Tanner, N.K., and Linder, P. (2006). The DEAD-box protein family of RNA helicases. Gene 367, 17-37. Cordin, O., Tanner, N.K., Doere, M., Linder, P., and Banroques, J. (2004). The newly discovered Q motif of DEAD-box RNA helicases regulates RNA-binding and helicase activity. The EMBO journal 23, 2478-2487. Gong, Z., Dong, C.H., Lee, H., Zhu, J., Xiong, L., Gong, D., Stevenson, B., and Zhu, J.K. (2005). A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis. The Plant cell 17, 256-267. Gorbalenya, A.E., Koonin, E.V. (1993). Helicases: amino acid sequence comparisons and structure–function relationships. Curr. Opin. Struct. Biol. 3, 419–429. Huang, C.K., Huang, L.F., Huang, J.J., Wu, S.J., Yeh, C.H., and Lu, C.A. (2010a). A DEAD-box protein, AtRH36, is essential for female gametophyte development and is involved in rRNA biogenesis in Arabidopsis. Plant & cell physiology 51, 694-706. Huang, T.S., Wei, T., Laliberte, J.F., and Wang, A. (2010b). A host RNA helicase-like protein, AtRH8, interacts with the potyviral genome-linked protein, VPg, associates with the virus accumulation complex, and is essential for infection. Plant physiology 152, 255-266. Kant, P., Kant, S., Gordon, M., Shaked, R., and Barak, S. (2007). STRESS RESPONSE SUPPRESSOR1 and STRESS RESPONSE SUPPRESSOR2, two DEAD-box RNA helicases that attenuate Arabidopsis responses to multiple abiotic stresses. Plant physiology 145, 814-830. Kovalev, N., Barajas, D., and Nagy, P.D. (2012). Similar roles for yeast Dbp2 and Arabidopsis RH20 DEAD-box RNA helicases to Ded1 helicase in tombusvirus plus-strand synthesis. Virology 432, 470-484. Li, D., Liu, H., Zhang, H., Wang, X., and Song, F. (2008). OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress. Journal of experimental botany 59, 2133-2146. Li, X., Gao, X., Wei, Y., Deng, L., Ouyang, Y., Chen, G., Li, X., Zhang, Q., and Wu, C. (2011). Rice APOPTOSIS INHIBITOR5 coupled with two DEAD-box adenosine 5'-triphosphate-dependent RNA helicases regulates tapetum degeneration. The Plant cell 23, 1416-1434. Linder, P., and Fuller-Pace, F. (2015). Happy birthday: 25 years of DEAD-box proteins. Methods in molecular biology (Clifton, N.J.) 1259, 17-33. Linder, P., Lasko, P.F., Ashburner, M., Leroy, P., Nielsen, P.J., Nishi, K., Schnier, J., and Slonimski, P.P. (1989). Birth of the D-E-A-D box. Nature 337, 121-122. Liu, M., Shi, D.Q., Yuan, L., Liu, J., and Yang, W.C. (2010). SLOW WALKER3, encoding a putative DEAD-box RNA helicase, is essential for female gametogenesis in Arabidopsis. Journal of integrative plant biology 52, 817-828. Pause, A., and Sonenberg, N. (1992). Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. The EMBO journal 11, 2643-2654. Pause, A., Methot, N., Svitkin, Y., Merrick, W.C., and Sonenberg, N. (1994). Dominant negative mutants of mammalian translation initiation factor eIF-4A define a critical role for eIF-4F in cap-dependent and cap-independent initiation of translation. The EMBO journal 13, 1205-1215. Shinozaki, K., Yamaguchi-Shinozaki, K., and Seki, M. (2003). Regulatory network of gene expression in the drought and cold stress responses. Current opinion in plant biology 6, 410-417. Stonebloom, S., Burch-Smith, T., Kim, I., Meinke, D., Mindrinos, M., and Zambryski, P. (2009). Loss of the plant DEAD-box protein ISE1 leads to defective mitochondria and increased cell-to-cell transport via plasmodesmata. Proceedings of the National Academy of Sciences of the United States of America 106, 17229-17234. Svitkin, Y.V., Pause, A., Haghighat, A., Pyronnet, S., Witherell, G., Belsham, G.J., and Sonenberg, N. (2001). The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure. RNA (New York, N.Y.) 7, 382-394. Tanner, N.K. (2003). The newly identified Q motif of DEAD box helicases is involved in adenine recognition. Cell cycle (Georgetown, Tex.) 2, 18-19. Tanner, N.K., Cordin, O., Banroques, J., Doere, M., and Linder, P. (2003). The Q motif: a newly identified motif in DEAD box helicases may regulate ATP binding and hydrolysis. Molecular cell 11, 127-138. Xiong, L., Schumaker, K.S., and Zhu, J.K. (2002). Cell signaling during cold, drought, and salt stress. The Plant cell 14 Suppl, S165-183. Xu, J., Liu, C., Li, M., Hu, J., Zhu, L., Zeng, D., Yang, Y., Peng, Y., Ruan, B., Guo, L., and Li, H. (2015). A rice DEAD-box RNA helicase protein, OsRH17, suppresses 16S ribosomal RNA maturation in Escherichia coli. Gene 555, 318-328. Yamaguchi-Shinozaki, K., and Shinozaki, K. (2006). Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual review of plant biology 57, 781-803. Zhu, J.K. (2002). Salt and drought stress signal transduction in plants. Annual review of plant biology 53, 247-273.
