大豆（Glycine max）GmDREB5互作蛋白GmUBC13的特性及功能

doi:10.3864/j.issn.0578-1752.2014.18.002

中国农业科学

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

大豆（Glycine max）GmDREB5互作蛋白GmUBC13的特性及功能

徐东北^1,2，于月华³，韩巧玲²，马亚男^1,2，高世庆⁴，田野²，徐兆师²，李连城²，曲延英³，马有志²，陈明²，陈耀锋¹

¹西北农林科技大学农学院，陕西杨凌 712100；
²中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程/农业部麦类生物学与遗传育种重点开放实验室，北京 100081；
³新疆农业大学农学院，乌鲁木齐 830052；
⁴北京杂交小麦工程技术研究中心，北京100097

收稿日期:2014-03-12 修回日期:2014-05-14 出版日期:2014-09-16 发布日期:2014-09-16
通讯作者: 通信作者陈耀锋，Tel：13060391299；E-mail：chenf3828@126.com 通信作者陈明，Tel：010-82108750；E-mail：chenming02@caas.cn
作者简介:徐东北，Tel：010-82108750；E-mail：xudongbei2006@126.com。
基金资助:
转基因生物新品种培育科技重大专项（2013ZX08002-002、2013ZX08002-005）

Characteristics and Function of a GmDREB5-Interacting Protein GmUBC13 in Soybean

XU Dong-bei^1,2, YU Yue-hua³, HAN Qiao-ling², MA Ya-nan^1,2, GAO Shi-qing⁴, TIAN Ye², XU Zhao-shi², LI Lian-cheng², QU Yan-ying³, MA You-zhi², CHEN Ming², CHEN Yao-feng¹

¹College of Agronomy, Northwest A &F University, Yangling 712100, Shaanxi;
²Institute of Crop Sciences, Chinese Academy of Agricultural Sciences/National Key Facility for Crop Gene Resources and Genetic Improvement/Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Beijing 100081;
³College of Agronomy, Xinjiang Agricultural University, Urumqi 830052;
⁴Beijing Hybrid Wheat Engineering and Technology Research Center, Beijing 100097

Received:2014-03-12 Revised:2014-05-14 Online:2014-09-16 Published:2014-09-16

摘要/Abstract

摘要： 【目的】鉴定大豆抗逆相关转录因子GmDREB5的互作蛋白，分析其互作蛋白GmUBC13的特性及其生物学功能，解析GmDREB5提高植物抗逆性的分子机制。【方法】通过酵母双杂交系统，以大豆GmDREB5的AP2功能域为诱饵对干旱处理的大豆cDNA文库进行筛选，获得GmDREB5候选互作蛋白后通过酵母互作及体外Pull-down试验确定GmDREB5与候选蛋白之间的互作关系；同时，分析互作蛋白GmUBC13的进化关系、蛋白结构及亚细胞定位等特性；通过半定量RT-PCR分析其互作蛋白GmUBC13在干旱、高盐、低温等非生物胁迫和激素ABA处理下的表达谱；通过转化烟草鉴定GmUBC13的生物学功能。【结果】通过筛选大豆干旱处理的cDNA文库获得一个GmDREB5互作蛋白GmUBC13（ubiquitin conjugating enzyme 13），GmUBC13属于泛素结合酶蛋白家族，GmUBC13含有UBCc保守域（ubiquitin-conjugating enzyme catalytic domain）、与泛素连接酶E3互作的氨基酸残基以及高度保守的半胱氨酸催化位点。进化树分析表明，GmUBC13的氨基酸序列与拟南芥（Arabidopsis thaliana）含有16个成员的E2家族的第XV亚组的AtUBC13A、AtUBC13B以及水稻（Oryza sativa）的泛素结合酶蛋白Os01g0673600分别具有99%、97%和97%的同源性。酵母互作试验及体外Pull-down分析证明GmUBC13与GmDREB5蛋白之间存在相互作用。表达特性分析表明，GmUBC13受干旱、高盐、低温等非生物胁迫和激素ABA处理的诱导表达。GmUBC13在ABA的胁迫条件下，1 h开始有表达，10 h时表达量上升到最大，24 h稍微降低；在干旱和盐胁迫条件下，1 h开始表达，并随着胁迫时间的增长，表达量逐渐上升，24 h表达量达到最大；在低温胁迫条件下，GmUBC13受诱导较快，5 h表达达到最大，在10 h和24 h时未表达。蛋白亚细胞定位结果显示，GmDREB5蛋白定位在细胞核和细胞膜上，GmUBC13定位在细胞核中。基因功能鉴定结果证明，过表达GmUBC13的转基因株系GmUBC13-1、GmUBC13-2和受体对照W38的幼苗在正常MS培养基上的生长状态基本相似，在不同浓度PEG胁迫条件下，转基因株系GmUBC13-1、GmUBC13-2和W38烟草叶片叶绿素含量都降低，根长和地上部生长都受到抑制，而转GmUBC13烟草的叶片叶绿素含量降低缓慢，转基因烟草各株系的根长、地面长度均高于对照W38，且在8% PEG处理条件下，转基因株系GmUBC13-2的叶绿素含量、根长及地面长度与W38的差异达极显著。将生长2周的转基因烟草株系GmUBC13-1、GmUBC13-2与W38移至营养土中控水处理21 d，复水处理6 d后显示，转基因烟草株系生长情况明显优于非转基因对照W38，且转基因株系的存活率显著高于对照W38，表明在烟草中过表达大豆GmUBC13可以显著提高转基因烟草的抗旱性。【结论】大豆泛素连接酶GmUBC13与抗逆相关转录因子GmDREB5互作，GmUBC13受各种非生物胁迫处理诱导表达，在烟草中过表达可以显著提高植物的抗旱性。

关键词: 大豆, DREB类转录因子基因, 泛素连接酶, 蛋白质互作, 基因功能

Abstract: 【Objective】The objectives of this research are to identify the interacting proteins of stress related DREB like transcription factor, GmDREB5 in soybean (Glycine max), analyze the characteristics and biological function of its interaction protein, GmUBC13, and study the regulation mechanism that GmDREB5 enhanced the stress tolerance in plants. 【Method】 By yeast two hybrid system, AP2 domain of GmDREB5 was used as a bait to screen the interacting proteins of GmDREB5 from the drought-treated soybean cDNA library, and further interaction between GmDREB5 and its candidate interacting protein was confirmed through interacting testing in yeast and pull-down assay in vitro, then, the properties of interacting protein, GmUBC13, such as phylogenetic tree, protein structure, subcellular localization were analyzed; expression profiles of GmUBC13 were tested under drought, salt, cold and ABA treatment by RT-PCR, and biological functions of GmUBC13 were also identified through transformation of tobacco in this research. 【Result】A GmDREB5-interacting protein, GmUBC13, belonging to the ubiquitin conjugating enzyme protein family, was obtained via screening drought-treated soybean cDNA library, and GmUBC13 contained UBCc (Ubiquitin-conjugating enzyme catalytic domain) domain, highly conserved E3 interaction residues and cysteine catalytic sites. Phylogenetic analyses showed that GmUBC13 shared the high homology with two members of the XV subgroup (containing total sixteen members) of ubiquitin conjugating enzyme like protein family, AtUBC13A (99%) and AtUBC13B (97%) in Arabidopsis and a ubiquitin conjugating enzyme like protein, Os01g0673600 (97%) in rice (Oryza sativa). Interaction between GmDREB5 and GmUBC13 was confirmed by interaction test in yeast and pull-down assay in vitro. The expression of GmUBC13 gene in soybean was analyzed by drought, high salt, low temperature and ABA treatment. After ABA application, the transcript of GmUBC13 was induced after 1 h and reached the peak at 10 h, and slight down-regulated at 24 h. Under drought- and salt-treatment, the transcript of GmUBC13 was induced after 1 h and gradually increased along with the treatment time, and reached the peak at 24 h; under the low temperature, the expression of GmUBC13 induced rapidly and reached the peak at 5 h, and there was no expression at 10 h and 24 h. Further subcellular localization analysis showed that GmDREB5 was located on the nuclear and cytoplasm, and GmUBC13 was located on the nuclear. The analysis result of gene function showed that there was no difference among transgenic tobacco lines GmUBC13-1, GmUBC13-2 and wild type (W38) under normal MS medium, whereas exposed to different concentrations of PEG, the chlorophyll contents decreased, and the length of root and segment above ground were inhibited in all plants. However, the chlorophyll contents were higher, and the length of root and segment above ground was longer in GmDREB13 transgenic plants compared with W38, and there was a significant difference between transgenic lines GmUBC13-2 and W38 under treatment of 8% PEG. For the drought tolerance assay, two-week old transgenic plants and W38 were transferred into soil and withheld water for 21 days, and then re-watered for 6 days. Final results showed that transgenic lines grew healthier than W38, and the survival rates of transgenic lines were significantly higher than W38, suggesting that overexpression of GmUBC13 enhanced drought tolerance in transgenic plant.【Conclusion】 Soybean ubiquitin conjugating enzyme, GmUBC13 interacted with stress related transcription factor GmDREB5, and GmUBC13 was induced by various stress treatments, and overexpression of GmUBC13 enhanced drought tolerance in transgenic tobacco plants.

Key words: soybean, DREB like transcription factor gene, ubiquitin-conjugating enzyme, interacting protein, gene function

徐东北，于月华，韩巧玲，马亚男，高世庆，田野，徐兆师，李连城，曲延英，马有志，陈明，陈耀锋. 大豆（Glycine max）GmDREB5互作蛋白GmUBC13的特性及功能[J]. 中国农业科学, 2014, 47(18): 3534-3544.

XU Dong-bei, YU Yue-hua, HAN Qiao-ling, MA Ya-nan, GAO Shi-qing, TIAN Ye, XU Zhao-shi, LI Lian-cheng, QU Yan-ying, MA You-zhi, CHEN Ming, CHEN Yao-feng. Characteristics and Function of a GmDREB5-Interacting Protein GmUBC13 in Soybean[J]. Scientia Agricultura Sinica, 2014, 47(18): 3534-3544.

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大豆（Glycine max）GmDREB5互作蛋白GmUBC13的特性及功能

Characteristics and Function of a GmDREB5-Interacting Protein GmUBC13 in Soybean

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