东莞大蕉超表达拟南芥CBF1基因及其抗寒性检测

doi:10.3864/j.issn.0578-1752.2012.08.022

摘要/Abstract

摘要： 【目的】研究超表达拟南芥CBF1基因（AtCBF1）对大蕉抗寒性的影响，为从大蕉克隆抗寒相关基因奠定基础。【方法】采用农杆菌介导法转化东莞大蕉的胚性细胞悬浮系，获得转AtCBF1基因的大蕉植株；利用GUS组织染色、PCR、RT-PCR以及RT-qPCR对转基因植株进行鉴定；比较低温处理后的转基因株系和对照的冷害特征以及超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量等生理生化指标，鉴定转基因大蕉植株的抗寒能力。【结果】试验共获得6个抗性再生转化系。GUS组织染色结果表明，除T1外，其余均为阳性；PCR鉴定结果表明，AtCBF1在6个抗性再生转化系均为阳性，而GUS基因在T1转化系中没有检测出；RT-PCR结果表明，AtCBF1在6个转化系均得到表达，对T1、T2和T3 3个转化系进行RT-qPCR检测发现，AtCBF1基因在3个转基因株系表达水平存在差异；在低温处理下，转基因植株的叶片相对电导率、MDA的累积都低于非转基因植株，而SOD总活性高于对照；低温处理条件下，转基因植株叶片的冷害症状明显轻于对照。【结论】AtCBF1在大蕉中超表达，具有增强大蕉SOD活性，降低因低温导致的MDA含量和离子渗漏率，缓解质膜过氧程度，进而改善大蕉植株抗低温胁迫的能力。

关键词: 大蕉(Musa spp.ABB group), 拟南芥CBF1基因, 转基因, 抗寒性

Abstract: 【Objective】To elucidate the effects of over-expression of AtCBF1 gene on tolerance to low-temperature in Musa ABB cv. Dongguandajiao and establish a foundation for cloning cold resistance relative genes from Dongguandajiao.【Method】Transgenic plants transformed by AtCBF1 gene were generated based on Agrobacterium tumerficiens-mediated transformation of embryogenic cell suspensions (ECS) of Dongguandajiao. The transgenic plants were confirmed via GUS staining, PCR, RT-PCR and real-time fluorescence quantitative RT-PCR(RT-qPCR). The cold tolerance of transgenic plants were evaluated by the comparisons of plant morphology and physiological parameters such as chilling injury symptom, superoxide dismutase (SOD) activities, malonaldehyde (MDA) content and so on among the transgenic lines and non-transformed plants (CK) after low-temperature treatment.【Result】Six resistant lines were initiated in this experiment. Results from GUS staining showed that all resistant plants were positive except T1 line. PCR showed that AtCBF1 gene was detected in the six transformed lines, but gus gene not in T1 line. Expression of AtCBF1 gene in the six transgenic lines was confirmed using RT-PCR. Difference existed among T1, T2 and T3 for AtCBF1 expression in RT-qPCR analysis. After low-temperature stress treatment, the relative electric conductivity and MDA content of leaves of transgenic lines were lower than that of non-transformed plants. However, the superoxide dismutase (SOD) activities was higher in transgenic lines. The chilling injury symptom of leaves of transgenic lines were much slighter than that of the CK under low-temperature treatment.【Conclusion】 Over-expression of AtCBF1 gene increased SOD activities and decreased MDA content and electrolyte leakage rate caused by low -temperature. Thus the degree of over-oxidation of the cellular membrane is lessenned and the low-temperature stress tolerant capacities in transgenic plantain plants are improved.

Key words: Musa spp. ABB group, AtCBF1 gene, transgenic, cold resistance

刘凯, 胡春华, 杜发秀, 张玉娥, 魏岳荣, 易干军. 东莞大蕉超表达拟南芥CBF1基因及其抗寒性检测[J]. 中国农业科学, 2012, 45(8): 1653-1660.

LIU Kai, HU Chun-Hua, DU Fa-Xiu, ZHANG Yu-E, WEI Yue-Rong, YI Gan-Jun. Over-Expression of the Arabidopsis CBF1 Gene in Dongguandajiao (Musa spp.ABB group) and Detection of Its Cold Resistance[J]. Scientia Agricultura Sinica, 2012, 45(8): 1653-1660.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.08.022

https://www.chinaagrisci.com/CN/Y2012/V45/I8/1653

参考文献

[1]Stockinger E J, Gilmour S J, Thomashow M F. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/ DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proceedings of the National Academy of Sciences of the USA, 1997, 94: 1035-1040.

[2]Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought and low temperature-responsive gene expression, respectively, in Arabidopsis. The Plant Cell, 1998, 10: 1391-1406.

[3]Haake V, Cook D, Riechmann J L, Pineda O, Thomashow M F, Zhang J Z. Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiology, 2002, 130: 639-648.

[4]Sakuma Y, Liu Q, Dubouzet J G, Abe H, Shinozaki K, Yamaguchi- Shinozaki K. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration and cold inducible gene expression. Biochemical and Biophysical Research Communications, 2002, 290(3): 998-1009.

[5]Jaglo-Ottosen K R, Gilmour S J, Zarka D G, Schabenberger O, Thomashow M F. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance. Science, 1998, 280: 104-106.

[6]Sharabi-Schwager M, Porat R, Samach A. Overexpression of the CBF2 transcriptional activator enhances oxidative stress tolerance in Arabidopsis plants. International Journal of Biology, 2011, 3(2): 94-105.

[7]Sharabi-Schwager M, Lers A, Samach A, Guy C L, Porat R. Overexpression of the CBF2 transcriptional activator in Arabidopsis delays leaf senescence and extends plant longevity. Journal of Experimental Botany, 2010, 61(1): 261-273.

[8]Zhao J S, Wei R, Zhi D Y, Wang L, Xia G M. Arabidopsis DREB1A/CBF3 bestowed transgenic tall fescue increased tolerance to drought stress. Plant Cell Report, 2007, 26: 1521-1528.

[9]Behnam B, Kikuchi A, Celebi-Toprak F, Kasuga M, Yamaguchi- Shinozaki K, Watanabe K N. Arabidopsis rd29A::DREB1A enhances freezing tolerance in transgenic potato. Plant Cell Report, 2007, 26: 1275-1282.

[10]Mi F G, Wang G H, Dong S J. Studies on the expression of exogenous CBF4 gene in transgenic wheatgrass. Sustainable use of Genetic Diversity in Forage and Turf Breeding, 2010, 6: 545-550.

[11]Singh S, Rathore M, Goyary D, Singh R K, Anandhan S, Sharma D K, Ahmed Z. Induced ectopic expression of At-CBF1 in marker-free transgenic tomatoes confers enhanced chilling tolerance. Plant Cell Report, 2011, 30: 1019-1028.

[12]Bendict A, Skinner J S, Meng R, Chang Y J, Bhalerao R, Huner N A, Finn C E, Chen T H, Hurry V. The CBF1-dependent low temperature signalling pathway, regulon and increase in freeze tolerance are conserved in Populus spp. Plant, Cell and Environment, 2006, 29: 1259-1272.

[13]Yang J S, Wang R, Meng J J, Bi Y P, Xu P L, Guo F, Wan S B, He Q W, Li X G. Overexpression of Arabidopsis CBF1 gene in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance. Journal of Plant Physiology, 2010, 167: 534-539.

[14]刘凯, 胡春华, 魏岳荣, 易干军, 邵秀红. 拟南芥CBF1基因植物表达载体构建及其对野生蕉的遗传转化.湖南农业大学学报:自然科学版, 2011, 37(3): 248-252.

Liu K, Hu C H, Wei Y R, Yi G J, Shao X H. Construction of plant expression vector containing CBF1 and its genetic transformation in wild banana. Journal of Hunan Agricultural University: Natural Sciences, 2011, 37(3): 248-252. (in Chinese)

[15]胡春华, 魏岳荣, 易干军, 黄秉智, 黄永红. 根癌农杆菌介导的香蕉高效遗传转化系统的建立.分子植物育种, 2010, 8(1): 172-178.

Hu C H, Wei Y R, Yi G J, Huang B Z, Huang Y H. Establishment of a high efficient Agrobacterium tumefaciens–mediated transformation system for banana. Molecular Plant Breeding, 2010, 8(1): 172-178. (in Chinese)

[16]Ghosh R, Paul S, Ghosh S K, Roy A. An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants. Journal of Virological Methods, 2009, 159: 34-39.

[17]Zhang Y J, Yang J S, Guo S J, Meng J J, Zhang Y L, Wan S B, He Q W, Li X G. Over-expression of the Arabidopsis CBF1 gene improves resistance of tomato leaves to low temperature under low irradiance. Plant Biology, 2011, 13(2): 362-367.

[18]Lee S C, Huh K W, An K, An G, Kim S R. Ectopic expression of a cold-inducible transcription factor, CBF1/DREB1b, in transgenic rice (Oryza sativa L.). Molecular Cell, 2004, 18(1): 107-114.

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

[20]Pino M T, Skinner J S, Jeknic Z, Hayes P M, Soeldner A H, Thomashow M F, Chen T H. Ectopic AtCBF1 over-expression enhances freezing tolerance and induces cold acclimation-associated physiological modi?cations in potato. Plant, Cell and Environment, 2008, 31: 393-406.

[21]Kasuga M, Miura S, Shinozaki K, amaguchi-Shinozaki K. A combination of the Arabidopsis DREB1A gene and stress-inducible rd29a promoter improved drought-and low-temperature stress tolerance in tobacco by gene transfer. Plant Cell Physiology, 2004, 45(3): 346-350.

[22]Welling A and Palva E T. Involvement of CBF Transcription Factors in Winter Hardiness in Birch. Plant Physiology, 2008, 147: 1199-1211.

[23]Kitashiba H, Ishizaka T, Isuzugawa K, Nishimura K, Suzuki T. Expression of a sweet cherry DREB1/CBF ortholog in Arabidopsis confers salt and freezing tolerance. Journal of Plant Physiology, 2004, 161: 1171-1176.

[24]Allen R D. Dissection of oxidative stress tolerance using transgenic plants. Plant Physiology, 1995, 107: 1049-1054.

[25]Clare D A, Rabinowitch H D, Fridovich I. Superoxide dismutase and chilling injury in Chlorella ellipsoidea. Archives of Biochemistry and Biophysics, 1984, 231(1): 158-163.

[26]Chu C C, Lee W C, Guo W Y, Pan S M, Chen L J, Li H, T Jinn S L. A copper chaperone for superoxide dismutase that confers three types of copper/zinc superoxide dismutase activity in Arabidopsis. Plant Physiology, 2005, 139: 425-436.

[27]Hodgson R A J, Raison J K. Lipid peroxidation and superoxide dismutase activity in relation to photoinhibition induced by chilling in moderate light. Planta, 1991, 185: 215-219.

[28]Wei S J, Sun Z Y, Han L, Yu L J. Transgenic tobacco plants over-expressing Arabidopsis transcriptional factor cbf1 show morphological and biochemical characteristics associated with cold tolerance. Asian Journal of Plant Sciences, 2006, 5(6): 932-939.

[29]李荣田, 张忠明, 张启发. RHL基因对粳稻的转化及转基因植株的耐盐性. 科学通报, 2002, 47: 613-617.

Li R T, Zhang Z M, Zhang Q F. Transformation of japonica rice with RHL gene and salt tolerance of transgenic plants. Chinese Science Bulletin, 2002, 47: 613-617. (in Chinese)

[30]吴关庭, 陈锦清, 胡张华, 郎春秀, 陈笑芸, 王伏林, 金卫, 夏英武. 根癌农杆菌介导转化获得耐逆性增强的高羊茅转基因植株. 中国农业科学, 2005, 38(12): 2395-2402.

Wu G T, Chen J Q, Hu Z H, Lang C X, Chen X Y, Wang F L, Jin W, Xia Y W. Production of transgenic tall fescue plants with enhanced stress tolerance by Agrobacterium tumefaciens-mediated transformation. Scientia Agricultura Sinica, 2005, 38(12): 2395-2402. (in Chinese)

[31]Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nature Biotechnology, 1999, 17: 287-291.