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【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。
【Objective】 The objective of this study was to identify the interacting proteins of GmDREB5, a transcription factor involved in soybean resistance, to analyze the characteristics of GmDC13 and its biological functions, and to elucidate the molecular mechanism of GmDREB5 in enhancing plant stress resistance. 【Method】 The yeast two-hybrid system was used to screen the arid-treated soybean cDNA library using the AP2 domain of soybean GmDREB5 as bait. GmDREB5 candidate interaction protein was obtained after yeast interaction and in vitro pull-down assay to determine the relationship between GmDREB5 and the candidate protein At the same time, we analyzed the evolutionary relationship, protein structure and subcellular localization of the interacting protein GmUBC13. The semi-quantitative RT-PCR analysis of its interaction protein GmUBC13 in drought, salt, low temperature and other abiotic stresses And hormone ABA treatment; identification of the biological function of GmUBC13 by tobacco transformation. 【Result】 A GmDREB5 interacting protein, GmUBC13 (ubiquitin conjugating enzyme 13), was obtained by screening arid soybean cDNA library. GmUBC13 belongs to the ubiquitin-conjugating enzyme family. GmUBC13 contains the ubiquitin-conjugating enzyme catalytic domain, Amino acid residues that prime ligase E3 interacts with and a highly conserved cysteine catalytic site. Phylogenetic tree analysis showed that the amino acid sequence of GmUBC13 shared 99% homology with AtUBC13A, AtUBC13B in Arabidopsis thaliana, an XV group of E2 family and Os01g0673600 in rice (Oryza sativa) , 97% and 97% homology. Yeast interaction test and in vitro Pull-down analysis showed that GmUBC13 interacts with GmDREB5 protein. Expression analysis showed that GmUBC13 was induced by ABA treatment under abiotic stresses such as drought, high salt and low temperature. Expression of GmUBC13 began at 1 h under ABA stress and reached a maximum at 10 h, slightly decreased at 24 h. Expression of GmUBC13 began at 1 h under drought and salt stress and was expressed as the stress time increased The amount of GmUBC13 was induced rapidly at 24 h and reached the maximum at 5 h, but not at 10 h and 24 h. The results of protein subcellular localization showed that GmDREB5 protein localized on the nucleus and cell membrane, and GmUBC13 was located in the nucleus. The results of gene function identification showed that the growth status of GmUBC13-1, GmUBC13-2 and W38 transgenic plants overexpressing GmUBC13 were similar in normal MS medium. Under the conditions of PEG stress, transgenic lines GmUBC13-1, GmUBC13-2 and W38 tobacco leaf chlorophyll content were reduced, root length and shoot growth were inhibited, and GmUBC13 tobacco leaf chlorophyll content decreased slowly, the root length of transgenic tobacco lines, the ground length are high Compared with W38, the difference of chlorophyll content, root length and ground length of transgenic line GmUBC13-2 with W38 under the conditions of 8% PEG treatment was extremely significant. The transgenic tobacco lines GmUBC13-1, GmUBC13-2 and W38, which had been growing for 2 weeks, were transferred to nutrient soil for 21 days. After rehydration for 6 days, the growth of transgenic tobacco lines was significantly better than that of non-transgenic controls W38 , And the survival rate of the transgenic lines was significantly higher than that of the control W38, indicating that overexpression of soybean GmUBC13 in tobacco can significantly improve the drought resistance of the transgenic tobacco. 【Conclusion】 Soybean ubiquitin ligase GmUBC13 interacts with anti-retrograde transcription factor GmDREB5. GmUBC13 is induced by various abiotic stress treatments. Overexpression in tobacco can significantly increase plant drought resistance.