独行菜抗冷相关基因的克隆及表达分析

doi:10.6048/j.issn.1001-4330.2018.04.012

新疆农业科学 ›› 2018, Vol. 55 ›› Issue (4): 689-696.DOI: 10.6048/j.issn.1001-4330.2018.04.012

独行菜抗冷相关基因的克隆及表达分析

张娜,胡昕,傅代辉,丁金鹏,李群

新疆大学生命科学与技术学院,乌鲁木齐 830046

收稿日期:2018-01-17 出版日期:2018-04-20 发布日期:2018-07-18
通信作者: 李群(1971-),女,河北人,副教授,博士,硕士生导师,研究方向为植物逆境生理与分子生物学,(E-mail)liqun_007@126.com
作者简介:张娜(1993-),女,甘肃白银人,硕士,研究方向为植物逆境生理与分子生物学,(E-mail)1558593931@qq.com
基金资助:
国家自然科学基金项目“新疆濒危植物高山离子芥抗冷相关功能基因组分析与关键基因克隆”(31160051);新疆维吾尔自治区科技厅科技支疆项目“新疆早春短命植物与模式植物拟南芥的比较基因组学与功能基因组学研究”(200840102-40)

Cloning and Expression Analysis of Cold Resistance Genes in Lepidium apetalum

ZHANG Na, HU Xin, FU Dai-hui, DING Jin-peng, LI Qun

College of Life Science and Technology, Xinjiang University, Urumuqi 830046, China

Received:2018-01-17 Online:2018-04-20 Published:2018-07-18
Correspondence author: Li Qun (1971-), female, Hebei, Ph.D., Master instructor, mainly engaged in research work in plant stress physiology and molecular biology, (E-mail) liqun_007@126.com
Supported by:
National Natural Science Foundation " Functional genome analysis of cold resistant and key gene clone in Xinjiang endangered plant Chorispora bungeana" (31160051); The Key Scientific Projects Xinjiang Uygur Autonomous Region " Comparative genomics and functional genomics of early spring ephemeral plants and model plants of Arabidopsis thaliana in Xinjiang" (200840102-40)

摘要/Abstract

摘要： 【目的】独行菜在种子萌发和幼苗生长阶段可耐受一定程度的低温胁迫,研究独行菜的低温耐受机制。【方法】设计兼并引物,从独行菜冷诱导叶片的cDNA中克隆获得LaCBF3和LaCOR15a基因核心片段。利用半定量RT-PCR法分析两个基因在不同胁迫处理下的表达情况。【结果】获得442 bp LaCBF3及405 bp LaCOR15a核心片段,经比对与拟南芥CBF3、COR15a基因核心片段相似性分别为84.38%、81.8%。半定量RT-PCR结果显示在独行菜幼苗中,LaCOR15a基因不仅响应低温胁迫,还能迅速响应高盐、干旱及ABA处理,不同条件下表达各有差异。LaCBF3仅对低温胁迫响应迅速。【结论】LaCBF3、LaCOR15a在独行菜幼苗响应低温胁迫的分子机制中具有重要的调控作用,且LaCOR15a基因也应答干旱、高盐等非生物胁迫。

关键词: 独行菜; 抗冷性 ; COR; CBF

Abstract: 【Objective】 Our previous studies of Lepidium apetalum Willd. showed that it could tolerate a certain degree of low temperature stress during germination and seedling growth. To further explore the mechanisms of cold tolerance in Lepidium apetalum, we cloned two key genes of cold resistance signal pathway named LaCBF3 and LaCOR15a and analyzed their expression patterns responded to abiotic stress.【Method】LaCBF3 and LaCOR15a genes were cloned by PCR method from cDNA of cold induced leaves of Lepidium plants using degenerate primers.【Result】The conservative regions of cloned 442 bp LaCBF3 and 405 bp LaCOR15a gene fragments had 84.38% and 81.8% similarity to Arabidopsis thaliana CBF3 gene and COR15a gene respectively. Semi quantitative RT-PCR showed that the LaCOR15a gene responded to low temperature stress as well as drought, salt and ABA treatment, while LaCBF3 only responded to low temperature stress rapidly in Lepidium seedlings. 【Conclusion】LaCBF3 and LaCOR15a play important regulatory roles in responding to low temperature in Lepidium apetalum during germination and seedling growth. Besides, LaCOR15a gene also responds to other abiotic stress such as drought, salt as well as ABA treatment.

Key words: Lepidium apetalum Willd; cold resistance; COR gene; CBF gene

中图分类号:

S188

张娜,胡昕,傅代辉,丁金鹏,李群. 独行菜抗冷相关基因的克隆及表达分析[J]. 新疆农业科学, 2018, 55(4): 689-696.

ZHANG Na, HU Xin, FU Dai-hui, DING Jin-peng, LI Qun. Cloning and Expression Analysis of Cold Resistance Genes in Lepidium apetalum[J]. Xinjiang Agricultural Sciences, 2018, 55(4): 689-696.

参考文献

[1] 赵慧, 赵一博, 郭江波,等. 植物耐受低温胁迫研究进展[J]. 种子, 2017, 36(5): 47-50.
   ZHAO Hui, ZHAO Yi-bo, GUO Jiang-bo, et al. (2017). Research Progress of Plant Under Low Temperature Stress [J]. Seed, 36(5): 47-50. (in Chinese)
[2] Jaglo-Ottosen, K. R., Gilmour, S. J., Zarka, D. G., Schabenberger, O., & Thomashow, M. F. (1998). Arabidopsis cbf1 overexpression induces cor genes and enhances freezing tolerance. Science, 280(5360): 104-106.
[3] Weiser, C. J. (1970). Cold resistance and injury in woody plants. Science, 169(3952): 1,269-1,278.
[4] Guy, C. L., Niemi, K. J., & Brambl, R. (1985). Altered gene expression during cold acclimation of spinach. Proc Natl Acad Sci U S A, 82(11): 3,673-3,677.
[5] 霍晨敏, 汤文强. 植物低温信号传导机制研究进展[J]. 生物技术通报, 2016, 32(10):27-33.
   HUO Chen-min, TANG Wen-qiang. (2016). A Review of Plant Cold Signal Transduction Mechanisms [J]. Biotechnology Bulletin, 32(10): 27-33. (in Chinese)
[6] Thomashow, M. F. (1994). Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance. Cold Spring Harbor Monograph Archive, 27(10): 807-834.
[7] Artus, N. N., Uemura, M., Steponkus, P. L., Gilmour, S. J., Lin, C., & Thomashow, M. F. (1996). Constitutive expression of the cold-regulated arabidopsis thaliana cor15a gene affects both chloroplast and protoplast freezing?tolerance. Proceedings of the National Academy of Sciences of the United States of America, 93(23): 13,404-13,409.
[8] Eric J. Stockinger, Sarah?J. Gilmour, & Michael?F. Thomashow. (1997). 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. Proc Natl Acad Sci Usa, 94(3): 1,035-1,040.
[9] Chinnusamy, V., Ohta, M., Kanrar, S., Lee, B. H., Hong, X., & Agarwal, M., et al. (2003). Ice1: a regulator of cold-induced transcriptome and freezing tolerance in arabidopsis. Genes Dev, 17(8):1,043-1,054.
[10] Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., & Yamaguchishinozaki, K., et al. (1998). 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. Plant Cell, 10(8): 1,391-1,406.
[11] 王俊峰, 孔维国, 张煜,等. 植物抗寒基因表达调控研究进展[J]. 西北植物学报, 2012, 32(4):852-858.
   WANG Jun-feng, KONG Wei-guo, ZHANG Yu, et al. (2012). Advance in Expression and Regulation of Cold-resistant Genes of Plants [J]. Acta Botanica Boreali-Occidentalia Sinica, 32(4): 852-858. (in Chinese)
[12] Jaglo-Ottosen, K. R., Gilmour, S. J., Zarka, D. G., Schabenberger, O., & Thomashow, M. F. (1998). Arabidopsis cbf1 overexpression induces cor genes and enhances freezing tolerance. Science, 280(5360): 104-106.
[13] White, T. C., Simmonds, D., Donaldson, P., & Singh, J. (1994). Regulation of bn115, a low-temperature-responsive gene from winter brassica napus. Plant Physiology, 106(3): 917-928.
[14] Monroy, A. F., Castonguay, Y., Laberge, S., Sarhan, F., Vezina, L. P., & Dhindsa, R. S. (1993). A new cold-induced alfalfa gene is associated with enhanced hardening at subzero temperature. Plant Physiology, 102(3): 873-879.
[15] Luo, X., Xiao, J., Wang, Z., Zhang, A., Tian, Y., & Wu, J. (2008). [overexpression of spinacia oleracea betaine aldehyde dehydrogenase (sobadh) gene confers the salt and cold tolerant in gossypium hirsutum l]. Chinese Journal of Biotechnology, 24(8): 1,464-1,469.
[16] Zhou, M., Wu, L., Liang, J., Shen, C., & Lin, J. (2012). Expression analysis and functional characterization of a novel cold-responsive gene cbcor15a from capsella bursa-pastoris. Molecular Biology Reports, 39(5): 5,169-5,179.
[17] Dunn, M. A., Brown, K., Lightowlers, R., & Hughes, M. A. (1996). A low-temperature-responsive gene from barley encodes a protein with single-stranded nucleic acid-binding activity which is phosphorylated in vitro. Plant Molecular Biology, 30(5): 947-959.
[18] Tsuda, K., Tsvetanov, S., Takumi, S., Mori, N., Atanassov, A., & Nakamura, C. (2000). New members of a cold-responsive group-3 lea/rab-related cor gene familyfrom common wheat (triticum aestivum l.). Genes & Genetic Systems, 75(4):179-188.
[19]丁金鹏, 张娜, 李群. 独行菜属植物种子总蛋白4种提取方法的比较[J]. 新疆农业科学, 2017, 54(7):1 305-1 312.
   DING Jin-peng, ZHANG Na, LI Qun. (2017). Comparative Study of Four Methods of Total Protein Extraction from Seeds of Two Species ofLepidiumPlants [J]. Xinjiang Agricultural Science, 54(7):1,305-1,312. (in Chinese)
[20] 李文明, 辛建攀, 魏驰宇,等. 植物抗寒性研究进展[J]. 江苏农业科学, 2017, 45(12):6-11.
   LI Wen-ming, XIN Jian-pan, WEI Chi-yu,et al. (2017). Research progress of plant cold resistance[J]. Jiangsu Agricultural Sciences,45(12):6-11.
[21] Zinn, K. E., Tuncozdemir, M., & Harper, J. F. (2010). Temperature stress and plant sexual reproduction: uncovering the weakest links. Journal of Experimental Botany, 61(7): 1,959-1,968.
[22] 徐呈祥. 提高植物抗寒性的机理研究进展[J]. 生态学报, 2012, 32(24):7 966-7 980.
   XU Cheng-xiang. (2012). Research progress on the mechanism of improving plant cold hardiness [J]. Acta Ecologica Sinica, 32(24): 7,966-7,980.(in Chinese)
[23] Medina, J., Bargues, M., Terol, J., P? Rez-Alonso, M., & Salinas, J. (1999). The arabidopsis cbf gene family is composed of three genes encoding ap2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration. Plant Physiology, 119(2): 463-470.
[24] Daie, J., & Campbell, W. F. (1981). Response of tomato plants to stressful temperatures : increase in abscisic acid concentrations. Plant Physiology, 67(1): 26-29.
[25] Liu, D., Li, W., Cheng, J., & Hou, L. (2014). Expression analysis and functional characterization of a cold-responsive gene cor15a, from arabidopsis thaliana. Acta Physiologiae Plantarum, 36(9): 2,421-2,432.
[26] Thalhammer, A., Bryant, G., Sulpice, R., & Hincha, D. K. (2014). Disordered cold regulated15 proteins protect chloroplast membranes during freezing through binding and folding, but do not stabilize chloroplast enzymes in vivo. Plant Physiology, 166(1): 190-201.
[27] Gilmour, S. J., Zarka, D. G., Stockinger, E. J., Salazar, M. P., Houghton, J. M., & Thomashow, M. F. (1998). Low temperature regulation of the arabidopsis CBF family of ap2 transcriptional activators as an early step in cold-induced cor gene expression. Plant Journal, 16(4): 433-442.
[28] Si, J., Wang, J. H., Zhang, L. J., Zhang, H., Liu, Y. J., & An, L. Z. (2009). Cbcor15, a cold-regulated gene from alpine chorispora bungeana, confers cold tolerance in transgenic tobacco. Journal of Plant Biology, 52(6): 593-601.
[29] Zhou, M., Wu, L., Liang, J., Shen, C., & Lin, J. (2012). Expression analysis and functional characterization of a novel cold-responsive gene cbcor15a from capsella bursa-pastoris. Molecular Biology Reports, 39(5): 5,169-5,179.
[30] Gilmour, S. J., Sebolt, A. M., Salazar, M. P., Everard, J. D., & Thomashow, M. F. (2000). Overexpression of the arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiology, 124(4): 1,854-1,865.
[31] Artus, N. N., Uemura, M., Steponkus, P. L., Gilmour, S. J., Lin, C., & Thomashow, M. F. (1996). Constitutive expression of the cold-regulated arabidopsis thaliana cor15a gene affects both chloroplast and protoplast freezing?tolerance. Proceedings of the National Academy of Sciences of the United States of America, 93(23): 13,404-13,409.
[32] Thalhammer, A., & Hincha, D. K. (2014). A mechanistic model of cor15 protein function in plant freezing tolerance: integration of structural and functional characteristics. Plant Signaling & Behavior, 9(12): e977722.

独行菜抗冷相关基因的克隆及表达分析

Cloning and Expression Analysis of Cold Resistance Genes in Lepidium apetalum

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