大豆GmHAT5的克隆及其转基因百脉根的抗盐分析

doi:10.3864/j.issn.0578-1752.2017.09.002

摘要/Abstract

摘要： 【目的】从大豆盐胁迫基因表达谱中筛选并克隆得到同源异型域亮氨酸拉链蛋白(HD-Zip)家族基因GmHAT5，将其转化豆科模式植物百脉根并进一步探究其抗盐调控机制。【方法】使用多种生物信息学软件对GmHAT5的开放读码框(ORF)、编码蛋白的分子量、等电点、序列结构和蛋白定位等进行预测，同时将大豆GmHAT5蛋白与其他10个物种的同源蛋白进行比对分析，并对GmHAT5在大豆不同器官及盐胁迫下的表达特性进行分析。此外，构建GmHAT5的植物超表达载体，通过对发根农杆菌LBA1334的转化，得到“复合体”百脉根植株，并在盐胁迫条件下对其进行抗盐表型分析;通过对根癌农杆菌EHA105的转化，获得百脉根的稳定转化株系, 并对其进行抗盐表型分析及相关生理指标检测。【结果】多种生物信息学软件分析表明，该片段包含1个1 038 bp的ORF，编码345个氨基酸。GmHAT5的理论分子量和等电点分别为39.17 kD和4.63。GmHAT5蛋白定位于细胞核，与其他HD-Zip家族蛋白一样，属于典型的核蛋白。序列分析表明，GmHAT5包含一个同源异型结构域和一个亮氨酸拉链结构域，属于第I类同源异型域亮氨酸拉链蛋白。同源蛋白比对表明其与野生大豆GsHAT5同源性最高。基因表达特性分析表明，GmHAT5在大豆植株的各个不同器官中均有表达，是一个受盐诱导上调表达的HD-Zip类转录因子。发状根转化的结果表明，使用200 mmol·L^-1 NaCl处理植株7 d后，“复合体”植株生长状态良好，对照组植株叶片明显萎蔫、失绿;不同NaCl浓度处理离体转基因发状根14 d后，对照组较试验组发状根明显干枯、生长受到抑制。稳定转化结果显示，不同盐浓度处理14 d后，转GmHAT5百脉根与两组对照植株相比生长状态更好。相关生理指标检测结果显示，与两组对照相比，转基因百脉根植株中丙二醛含量和相对质膜透性明显降低(P<0.05), 而叶绿素含量和根系活力则显著升高(P<0.05)。测定不同植株阳离子含量的结果表明，转基因百脉根株系与两组对照相比，Na⁺在叶片和根中含量显著下降，K⁺和Ca²⁺在叶片和根中含量显著升高。【结论】从大豆中克隆得到一个编码HD-ZipⅠ类同源异型域亮氨酸拉链蛋白基因GmHAT5，该基因在大豆中受盐胁迫诱导表达量显著升高。超量表达GmHAT5显著增强百脉根的耐盐能力，发状根转化法可以作为一种快速有效筛选抗盐候选基因的手段。推测GmHAT5在大豆盐胁迫应激调控中扮演重要角色。

关键词: 大豆, 同源异型域亮氨酸拉链蛋白(HD-Zip), 转录因子, 百脉根, 耐盐性

Abstract: 【Objective】Based on RNA-Seq profiling of the homeodomain leucine zipper protein (HD-Zip) transcription factor family in soybean (Glycine max) during salt stress, screening and cloning of a salt-induced gene (GmHAT5) from soybean were conducted. GmHAT5 was transformed into Lotus japonicus, the legume model system, to further understand the mechanism of salt tolerance. 【Method】The ORF of GmHAT5, the protein molecular weight, isoelectric point, sequence structure and protein localization were analyzed by some bioinformatics programs. Meanwhile, the homologous protein alignments with 10 other species, and relative expression levels of GmHAT5 in different organs and under saline stress were also analyzed. The overexpression vector of GmHAT5 was constructed and transformed into Agrobacterium rhizogenes strain LBA1334 to obtain the “composite” Lotus japonicus plants and the salt resistant phenotype was analyzed under salt stress. The overexpression vector of GmHAT5 was also transformed into Agrobacterium tumefaciens strain EHA105 to obtain the stable transgenic Lotus japonicus plants and then the phenotype and related physiological indexes were analyzed under salt stress. 【Result】Bioinformatics software analysis showed that the GmHAT5 contained an ORF with 1 038 bp, encoded 345 amino acid. The theoretical molecular weight and isoelectric point of GmHAT5 protein were 39.17 kD and 4.63, respectively. GmHAT5, located in the nucleus as other HD-Zip family proteins, was a typical nuclear protein. Sequence analysis showed that GmHAT5, which belonged to the first class of plant HD-Zip protein, contained a homeobox domain and a leucine zipper domain. The homologous protein alignments showed that GmHAT5 had a high similarity with GsHAT5. The hairy root transformation result showed that, after being treated with 200 mmol·L^-1 NaCl for 7 d, the “composite” plants grew well, while the empty vector control plants exhibited discoloration and stunted growth. Transgenic hairy roots in vitro culture was treated with a different concentration of NaCl for 14 d. Compared with the overexpressing transgenic hairy roots, the control group shriveled up and its growth was inhibited significantly. Stable transformation result showed that, after being treated with different concentration of NaCl for 14 d, the transgenic plants grew well compared to the two control groups. There were significantly changes in malondialdehyde content and relative membrane permeability caused by saline stress in transgenic plants compared to the two control groups (P<0.05). Moreover, transgenic plants had higher levels of chlorophyll content and root activity compared with the two control groups under saline stress conditions (P<0.05). Compared with the control plants, transgenic plants had lower levels of Na⁺ content in leaves and roots, while K⁺ and Ca²⁺ contents in leaves and roots increased significantly in transgenic plants.【Conclusion】 A HD-ZipⅠclass homeodomain leucine zipper protein gene (GmHAT5) was cloned from soybean, the expression level was significantly increased under salt stress. Over-expression of GmHAT5 gene could enhance resistance to saline in Lotus japonicus. Hairy root transformation method could be used as a quick effective means for screening of salt tolerance candidate gene. These imply that GmHAT5 may play an important role in salt stress regulation in soybean.

Key words: Glycine max, homeodomain leucine zipper protein (HD-Zip), transcription factor, Lotus japonicus, saline tolerance

柯丹霞，李祥永，王磊，程琳，刘永辉，李小艳，王慧芳. 大豆GmHAT5的克隆及其转基因百脉根的抗盐分析[J]. 中国农业科学, 2017, 50(9): 1559-1570.

KE DanXia, LI XiangYong, WANG Lei, CHENG Lin, LIU YongHui, LI XiaoYan, WANG HuiFang. Isolation of GmHAT5 from Glycine max and Analysis of Saline Tolerance for Transgenic Lotus japonicus[J]. Scientia Agricultura Sinica, 2017, 50(9): 1559-1570.

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