Article | . 2014 Vol. 32, Issue. 1
Characterization of a Cold Tolerance-related Gene, BrCSR, Derived from Brassica rapa



Department of Horticultural Biotechnology, Kyunghee University1




2014.. 91:99


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The objective of this study is to identify cold-tolerance genes in Brassica rapa. In order to acheive this goal, we analyzed a KBGP-24K oligo chip data [BrEMD (B. rapa EST and Microarray Database)] using B. rapa ssp. pekinensis inbred line ‘Chiifu’ under cold stress condition (4°C). Among 23,929 unigenes of B. rapa, 417 genes (1.7%) were primarily identified as cold responsive genes that were expressed over 5-fold higher than those of wild type control, and then a gene which has unknown function and has full length sequence was selected. It was named BrCSR (B. rapa Cold Stress Resistance). BrCSR was transformed using expression vector pSL101 to confirm whether BrCSR can enhance cold tolerance in tobacco plants. T1 transgenic tobacco plants expressing BrCSR were selected by PCR and Southern hybridization analyses, and the function of BrCSR was characterized by expression level analysis and phenotype observation under cold stress condition. The expression level of BrCSR in transgenic tobacco plants increased up to about two folds in quantitative real-time RT-PCR assay and this was very similar to Northern blot hybridization analysis. Analysis of phenotypic characteristics clearly elucidated that transgenic tobaccos expressing BrCSR were more cold tolerant than wild type control under 4°C treatment. Based on these results, we conclude that the over-expression of BrCSR might be closely related to the enhancement of cold tolerance.



1. Agarwal, M., Y. Hao, A. Kapoor, C.H. Dong, H. Fujii, X. Zheng, and J.K. Zhu. 2006. A R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. J. Biol. Chem. 281:37636-37645.  

2. Butt, A.J., C.M. Sergio, C.K. Inman, L.R. Anderson, C.M. McNeil, A.J. Russell, M. Nousch, T. Preiss, A.V. Biankin, R.L. Sutherland, and E.A. Musgrove. 2008. The estrogen and c-Myc target gene HSPC111 is over-expressed in breast cancer and associated with poor patient outcome. Breast Cancer Res. 10:R28.  

3. Desveaux, D., A. Marechal, and N. Brisson. 2005. Whirly tran-scription factors: Defense gene regulation and beyond. Trends Plant Sci. 10:95-102.   

4. Dubouzet, J.G., Y. Sakuma, Y. Ito, M. Kasuga, E.G. Dubouzet, S. Miura, M. Seki, K. Shinozaki, and K. Yamaguchi-Shinozaki. 2003. OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J. 33:751-763.  

5. Fowler, S. and M.F. Thomashow. 2002. Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 14:1675-1690.  

6. Foyer, C.H., H. Vanacker, L.D. Gomez, and J. Harbinson. 2002. Regulation of photosynthesis and antioxidant metabolism in maize leaves at optimal and chilling temperatures: Review. Plant Physiol. Biochem. 40:659-668.  

7. Ge, L.F., D.Y. Chao, M. Shi, M.Z. Zhu, J.P. Gao, and H.X. Lin. 2008. Overexpression of the trehalose-6-phosphate phosphatase gene OsTPP1 confers stress tolerance in rice and results in the activation of stress responsive genes. Planta 228:191-201.  

8. Gómez-Merino, F.C., C.A. Brearley, M. Ornatowska, M.E. Abdel- Haliem, M.I. Zanor, and B. Mueller-Roeber. 2004. AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phos-phorylates 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2- dioleoyl-sn-glycerol and exhibits cold-inducible gene expression. J. Biol. Chem. 279:8230-8241.  

9. Greer, D.H. 1990. The combined effects of chilling and light stress on photoinhibition of photosynthesis and its subsequent recovery. Plant Physiol. Biochem. 28:447-455.  

10. Guy, C., F. Kaplan, J. Kopka, J. Selbig, and D.K. Hincha. 2008. Metabolomics of temperature stress. Physiol. Plant 132:220-235.  

11. Ito, Y., K. Katsura, K. Maruyama, T. Taji, M. Kobayashi, M. Seki, K. Shinozaki, and K. Yamaquchi-Shinozaki. 2006. Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol. 47:141-153.  

12. Jaglo, K.R., S. Kleff, K.L. Amundsen, X. Zhang, V. Haake, J.Z. Zhang, T. Deits, and M.F. Thomashow. 2001. Components of the Arabidopsis C-repeat/dehydrationresponsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol. 127:910-917.  

13. Jen, C.H., I.W. Manfield, I. Michalopoulos, J.W. Pinney, W.G. Willats, P.M. Gilmartin, and D.R. Westhead. 2006. The Arabidopsis co-expression tool (ACT): A WWW-based tool and database for microarray-based gene expression analysis. Plant J. 46:336-348.  

14. Jiang, Y.Y., R.J. Chen, J.L. Dong, Z.J. Xu, and X.L. Gao. 2012. Analysis of GDSL lipase (GLIP) family genes in rice (Oryza sativa). Plant Omics 5:351-358.  

15. Kim, J.Y., S.J. Park, B. Jang, C.H. Jung, S.J. Ahn, C.H. Goh, K. Cho, O. Han, and H. Kang. 2007. Functional characterization of a glycine-rich RNA-binding protein 2 in Arabidopsis thaliana under abiotic stress conditions. Plant J. 50:439-451.  

16. Kundel, D.W., E. Stromquist, A.L. Greene, O. Zhdankin, R.R. Regal, and T.A. Rose-Hellekant. 2012. Molecular characterizations of Nop16 in murine mammary tumors with varying levels of c-Myc. Transgenic Res. 21:393-406.  

17. Lee, B.H., D.A. Henderson, and J.K. Zhu. 2005. The Arabidopsis cold-responsive transcriptome and its control by ICE1. Plant Cell 17:3155-3175.  

18. Lee, M.K., H.S. Kim, S.H. Kim, and Y.D. Park. 2004. Analysis of T-DNA integration patterns in transgenic tobacco plants. J. Plant Biol. 47:179-186.  

19. Lee, S.C., M.H. Lim, J.A. Kim, S.I. Lee, J.S. Kim, M. Jin, S.J. Kwon, J.H. Mun, Y.K. Kim, H.U. Kim, Y. Hur, and B.S. Park. 2008. Transcriptome analysis in Brassica rapa under the abiotic stresses using Brassica 24K oligo microarray. Mol. Cells 26:595-605.  

20. Lee, S.C., M.H. Lim, J.G. Yu, B.S. Park, and T.J. Yang. 2012. Genome-wide characterization of the CBF/DREB1 gene family in Brassica rapa. Plant Physiol. Biochem. 61:142-152.  

21. Oh, S.J., C.W. Kwon, D.W. Choi, S.I. Song, and J.K. Kim, 2007. Expression of barley HvCBF4 enhances tolerance to abiotic stress in transgenic rice. Plant Biotechnol. J. 5:646-656.  

22. Petricka, J.J. and T.M. Nelson. 2007. Arabidopsis nucleolin affects plant development and patterning. Plant Physiol. 144:173-186.  

23. Purugganan, M.M., J. Braam, and S.C. Fry. 1997. The Arabidopsis TCH4 xyloglucan endotransglycosylase. Substrate specificity, pH optimum, and cold tolerance. Plant Physiol. 115:181-190.  

24. Qin, F. Y. Sakuma, J. Li, Q. Liu, Y.Q. Li, K. Shinozaki, and K. Yamaguchi-Shinozaki. 2004. Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold- responsive gene expression in Zea mays L. Plant Cell. Physiol. 45:1042-1052.  

25. Rosado, A., E.J. Sohn, G. Drakakaki, S. Pan, A. Swidergal, Y. Xiong, B.H. Kang, R.A. Bressan, and N.V. Raikhel. 2010. Auxin- mediated ribosomal biogenesis regulates vacuolar trafficking in Arabidopsis. Plant Cell 22:143-158.  

26. Saijo, Y., S. Hata, J. Kyozuka, K. Shimamoto, and K. Izui. 2000. Over-expression of a single Ca-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J. 23:319-327.  

27. Savitch, L.V., A.G. Ivanov, L. Gudynaite-Savitch, N.P.A. Huner, and J. Simmonds. 2009. Effects of low temperature stress on excitation energy partitioning and photoprotection in Zea mays. Funct. Plant Biol. 36:37-49.  

28. Savitch, L.V., A.G. Ivanov, L. Gudynaite-Savitch, N.P.A. Huner, and J. Simmonds. 2011. Cold stress effects on PSI photochemistry in Zea mays: Differential increase of FQR-dependent cyclic electron flow and functional implications. Plant Cell Physiol. 52:1042-1054.  

29. Seki, M., M. Narusaka, J. Ishida, T. Nanjo, M. Fujita, Y. Oono, A. Kamiya, M. Nakajima, A. Enju, T. Sakurai, M. Satou, K. Akiyama, T. Taji, K. Yamaguchi-Shinozaki, P. Carninci, J. Kawai, Y. Hayashizaki, and K. Shinozaki. 2002. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high salinity stresses using a full-length cDNA microarray. Plant J. 31:279-292.  

30. Shou, H., P. Bordallo, and K. Wang. 2004. Expression of the nicotiana protein kinase (NPK1) enhanced drought tolerance in transgenic maize. J. Exp. Bot. 55:1013-1019.  

31. Takenaka, M., A. Zehrmann, D. Verbitskiy, M. Kugelmann, B. Härtel, and A. Brennicke. 2012. Multiple organellar RNA editing factor (MORF) family proteins are required for RNA editing in mitochondria and plastids of plants. Proc. Natl. Acad. Sci. USA 109:5104-5109.  

32. Vellosillo, T., M. Martínez, M.A. López, J. Vicente, T. Cascón, L. Dolan, M. Hamberg, and C. Castresana. 2007. Oxylipins produced by the 9-lipoxygenase pathway in Arabidopsis regulate lateral root development and defense responses through a specific signaling cascade. Plant Cell 19:831-846.  

33. Wang, W., B. Vinocur, and A. Altman. 2003. Plant responses to drought, salinity and extreme temperatures: Towards genetic engineering for stress tolerance. Planta 218:1-14.  

34. Wang, W., B. Vinocur, O. Shoseyov, and A. Altman. 2001. Bio-technology of plant osmotic stress tolerance: Physiological and molecular considerations. Acta Hort. 560:285-292.  

35. Winter, D., B. Vinegar, H. Nahal, R. Ammar, G.V. Wilson, and N.J. Provart. 2007. An “electronic fluorescent pictograph” browser for exploring and analyzing large-scale biological data sets. PLoS ONE 2:e718.  

36. Xiang, Y., Y. Huang, and L. Xiong. 2007. Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement. Plant Physiol. 144:1416-1428.  

37. Yang, K.A., C.J. Lim, J.K. Hong, Z.L. Jin, J.C. Hong, D.J. Yun, W.S. Chung, S.Y. Lee, M.J. Cho, and C.O. Lim. 2005. Identification of Chinese cabbage genes upregulated by prolonged cold by using microarray analysis. Plant Sci. 168:959-966.  

38. Yokoi, S., S.I. Higashi, S. Kishitani, N. Murata, and K. Toriyama. 1998. Introduction of the cDNA for shape Arabidopsis glycerol- 3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acids and chilling tolerance of photosynthesis on rice. Mol. Breed. 4:269-275.  

39. Zhang, Y., T.W. Yang, L.J. Zhang, T.G. Zhang, C.X. Di, S.J. Xu, and L.Z. An. 2006. Isolation and expression analysis of two cold-inducible genes encoding putative CBF transcription factors from Chinese cabbage (Brassica pekinensis Rupr.). J. Integr. Plant Biol. 48:848-856.