欧美杨锌指蛋白转录因子基因（ZxZF）的遗传转化及抗旱性初步分析

doi:10.11707/j.1001-7488.20140305

摘要/Abstract

摘要：

C2H2型锌指蛋白转录因子在激活胁迫相关基因、增强植物抗逆性方面具有重要的作用。以来自极端抗逆木本植物霸王C2H2型锌指蛋白转录因子基因（ZxZF）为外源基因，利用农杆菌介导法对欧美杨‘渤丰1号’进行遗传转化。经卡那霉素筛选获得80株抗性植株，通过PCR检测获得17株呈阳性植株，Southern印记杂交检测证实外源基因以单拷贝方式整合到5个转基因株系体内，RT-PCR及qRT-PCR分子检测表明，外源基因在4个转基因株系中成功表达。PEG模拟干旱胁迫试验结果初步表明，转基因株系叶片相对含水量和脯氨酸含量均显著高于非转基因植株10%以上，转基因株系抗旱性得到一定程度提高。研究表明ZxZF转录因子基因对提高转基因杨树抗旱能力具有重要作用，在培育抗旱林木优良品种中具有重要育种价值。

关键词: 欧美杨, 锌指蛋白基因, 遗传转化, 抗旱性

Abstract:

The C2H2 zinc finger protein transcription factor plays an important role in the activation of stress-related genes, and increase in plant resistance. In this study, an C2H2 zinc finger protein transcription factor (ZxZF) from shrub Sarcozygium xanthoxylon with strong drought tolerance was used as exogenous gene,and the Agrobacterium-mediated method was applied for transgenic transformation to an elite hybrid clone of Populus × euramericana cl.‘Bofeng1’. A total 80 resistant plants were gained via strict selection by kanamycin. Among them 17 positive transgenic plants were verified by PCR analysis, and five transgenic plants were finally confirmed with one copy of the exogenous gene by Southern blot analysis. Four of these clones were verified over-expression of ZxZF by RT-PCR and qRT-PCR analysis. Under the drought stress conditions, transgenic clones showed more than 10% significantly higher of relative water content (RWC) and accumulation of proline content, compared with non-transgenic control plants. These results showed an improved tolerance to drought stress in transgenic poplar. Our study suggested that ZxZF transcription factor played a crucial role in transgene poplar response to drought stress and was useful in developing trees with enhanced tolerance to drought.

Key words: Populus×euramericana, zinc-finger protein gene, genetic transformation, drought resistance

中图分类号:

张伟溪, 刘浡洋, 丁昌俊, 张冰玉, 黄秦军, 褚延广, 苏晓华. 欧美杨锌指蛋白转录因子基因（ZxZF）的遗传转化及抗旱性初步分析[J]. 林业科学, 2014, 50(3): 31-37.

Zhang Weixi, Liu Boyang, Ding Changjun, Zhang Bingyu, Huang Qinjun, Chu Yanguang, Su Xiaohua. Transformation of Zinc Finger Protein Transcription Factor Gene (ZxZF) and Preliminary Evaluation of Drought Tolerance in Populus × euramericana[J]. Scientia Silvae Sinicae, 2014, 50(3): 31-37.

参考文献

崔旭东, 苏晓华, 张冰玉, 等. 2012. 欧美杨渤丰1号高效组培再生体系建立. 林业科学研究, 25(2): 157-162.
(Cui X D, Su X H, Zhang B Y, et al. 2012. Establishment of an efficient regeneration System of Populus × euramericana cl.'Bofeng 1'. Forest Research, 25(2): 157-162. [in Chinese] )
李合生. 2007. 植物生理生化实验原理和技术. 北京: 高等教育出版社, 164, 137-137, 258-260.
李义良, 苏晓华, 张冰玉, 等. 2007. 外源SacB基因在银腺杂种杨基因组中的表达及抗旱性分析. 北京林业大学学报, 29(2): 1-6.
(Li Y L, Su X H, Zhang B Y, et al. 2007. Molecular detection and drought tolerance of SacB-transgenic Populus alba×P. glandulosa. Journal of Beijing Forestry University, 29(2): 1-6. [in Chinese] )
苏晓华, 张冰玉,黄烈健, 等. 2003.转基因林木研究进展. 林业科学研究, 16(1): 95-103.
(Su X H, Zhang B Y, Huang L J, et al. 2003. Forest Research, Genetic engineering of forest trees. Forest Research, 16(1): 95-103. [in Chinese] )
萨姆布鲁克 J, 拉塞尔 D W. 2002. 分子克隆实验指南.3版. 北京:科学出版社.
(Sambrook J, Russell D W. 2002. Molecular cloning: A laboratory manual, 2nd Ed. Beijing: Science Press. [in Chinese] )
王俊英, 尹伟伦, 夏新莉. 2005. 胡杨锌指蛋白基因克隆及其结构分析. 遗传, 27(2): 245-248.
(Wang J Y, Yin W L, Xia L L. 2005. Cloning and structure analysis of zinc finger protein gene in Populus euphratica oliver. Hereditas(Beijing), 27(2): 245-248. [in Chinese] )
严安心, 谢瑜, 吉柔风, 等. 2012. 一个麻疯树RING型锌指蛋白基因JcRFP 1 的克隆及表达. 植物生理学报, 48(5): 456-464.
(Yan A X, Xie Y, Ji R F, et al. 2012. Cloning and expression of a RING finger protein gene JcRFP1 from Jatropha curcas L. Plant Physiology Journal, 48(5): 456-464. [in Chinese] )
杨金水, 王光清. 1995. 转基因的失活与沉默. 生物工程进展, 15 (3) :41-45.
张冰玉, 苏晓华, 黄秦军, 等. 2005. 转果聚糖蔗糖转移酶基因银腺杨的获得(英文). 林业科学, 41(3): 48-53.
(Zhang B Y, Su X H, Huang Q J, et al. 2005. Regeneration of transgenic poplar(Populus alba×P.glandulosa) expressing levansucrase from bacillus subtilis. Scientia Silvae Sinicae, 41(3): 48-53. [in Chinese] )
中国科学院中国植物志编辑委员会. 1998. 中国植物志(第43卷, 第一分册). 北京: 科学出版社, 140-142.
(Editorialcommittee of Flora of China. 1998. Flora reipublicae popularis sinicae(Vol.43 Book 1). Beijing: Science Press, 140-142. [in Chinese] )
朱家红, 李辉亮, 屠发志, 等. 2006. 巴西橡胶树环锌指蛋白基因克隆及其分子特性(英文). 植物生理与分子生物学学报, 32(6): 627-633.
(Zhu J H, Li H L, Tu F Z, et al. 2006. Cloning and molecular characterization of a RING Zinc-Finger Gene of Hevea brasiliensis. Journal of Plant Physiology and Molecular Biology, 32(6): 627-633. [in Chinese] )
An Y, Wang Y, Lou L, et al. 2011. A novel zinc-finger-like gene from Tamarix hispida is involved in salt and osmotic tolerance. J Plant Res, 124(6):689-97.
Castonguay Y, Markhart A H. 1992. Leaf gas exchange in water-stressed common bean and tepary bean. Crop Sci, 32(4): 980-986.
Davletova S, Schlauch K, Coutu J, et al. 2005. The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in Arabidopsis. Plant Physiology, 139(2): 847-856.
Englbrecht C C, Schoof H, Böhm S. 2004. Conservation, diversification and expansion of C2H2 zinc finger proteins in the Arabidopsis thaliana genome. BMC Genomics, 5(1):39.
Hsieh T, Lee J, Charng Y, et al. 2002a. Tomato plants ectopically expressing Arabidopsis CBF 1 show enhanced resistance to water deficit stress. Plant Physiology, 130(2): 618-626.
Hsieh T, Lee J, Yang P, et al. 2002b. Heterology expression of the Arabidopsis C-Repeat/Dehydration Response Element Binding Factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato. Plant Physiology, 129(3): 1086-1094.
Huang J, Sun S, Xu D, et al. 2009. Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245. Biochemical and Biophysical Research Communications, 389(3): 556-561.
Jain M, Tyagi A K, Khurana J P. 2008. Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants. Plant Cell Reports, 27(4): 767-778.
Kim J C, Lee S H, Cheong Y H, et al. 2001. A novel cold-inducible zinc finger protein from soybean, SCOF-1, enhances cold tolerance in transgenic plants. The Plant Journal, 25(3): 247-259.
Li Y, Su X, Zhang B, et al. 2009. Expression of jasmonic ethylene responsive factor gene in transgenic poplar tree leads to increased salt tolerance. Tree Physiology, 29(2): 273-279.
Mittler R, Kim Y, Song L, et al. 2006. Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress. FEBS Letters, 580(28): 6537-6542.
Mukhopadhyay A, Vij S, Tyagi A K. 2004. Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco. Proceedings of the National Academy of Sciences of the United States of America, 101(16): 6309-6314.
Nanjo T, Kobayashi M, Yoshiba Y, et al. 1999. Biological functions of proline in morphogenesis and osmotolerance revealed in antisense transgenic Arabidopsis thaliana. Plant J, 18(2): 185-193.
Ravikumar R L, Patil B S, Salimath P M. 2003. Drought tolerance in sorghum by pollen selection using osmotic stress. Euphytica, 133(3): 371-376.
Sakamoto H, Araki T, Meshi T, et al. 2000. Expression of a subset of the Arabidopsis Cys2/His2-type zinc-finger protein gene family under water stress. Gene, 248(1): 23-32.
Sakamoto H, Maruyama K, Sakuma Y, et al. 2004. Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiology, 136(1): 2734-2746.
Sinclair T R, Ludlow M M. 1986. Influence of soil water supply on the plant water balance of four tropical grain legumes. Australian Journal of Plant Physiology, 13(3): 329-341.
Sugano S, Kaminaka H, Rybka Z, et al. 2003. Stress-responsive zinc finger gene ZPT 2-3 plays a role in drought tolerance in petunia. The Plant Journal, 36(6): 830-841.
Takatsuji H. 1998. Zinc-finger transcription factors in plants. Cellular and Molecular Life Sciences CMLS, 54(6): 582-596.
Vogel J T, Zarka D G, Van Buskirk H A, et al. 2005. Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis. The Plant Journal, 41(2): 195-211.
Xu S, Wang X, Chen J. 2007. Zinc finger protein 1 (ThZF1) from salt cress (Thellungiella halophila) is a Cys-2/His-2-type transcription factor involved in drought and salt stress. Plant Cell Reports, 26(4): 497-506.
Yamaguchi-Shinozaki K, Shinozaki K. 1994. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. The Plant Cell Online, 6(2): 251-264.
Yang X, Sun C, Hu Y, et al. 2008. Molecular cloning and characterization of a gene encoding RING zinc finger ankyrin protein from drought-tolerant Artemisia desertorum. Journal of Biosciences, 33(1): 103-112.
Zhu Z, Shi J, Xu W, et al. 2013. Three ERF transcription factors from Chinese wild grapevine Vitis pseudoreticulata participate in different biotic and abiotic stress-responsive pathways. J Plant Physiol, 170(10): 923-933.
Zou M, Guan Y, Ren H, et al. 2008. A bZIP transcription factor, OsABI5, is involved in rice fertility and stress tolerance. Plant Molecular Biology, 66(6): 675-683.

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