大豆GmHAT5的克隆及其转基因百脉根的抗盐分析

发布时间：2018-02-25 23:03

本文关键词： 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　出处：《中国农业科学》2017年09期 　论文类型：期刊论文

【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 k D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(P0.05),而叶绿素含量和根系活力则显著升高(P0.05)。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Ca~(2+)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。
[Abstract]:[objective] to screen and clone the homologous gene GmHAT5 of leucine zipper HD-Zipprotein family from soybean salt stress gene expression profile, to transform GmHAT5 into the legume model plant, and to further explore its salt-resistance regulation mechanism. Using a variety of bioinformatics software, the open reading frame of GmHAT5, encoding the molecular weight of the protein, The isoelectric point, sequence structure and protein location were predicted. At the same time, the soybean GmHAT5 protein was compared with the homologous proteins of other 10 species, and the expression characteristics of GmHAT5 in different organs and salt stress of soybean were analyzed. The plant superexpression vector of GmHAT5 was constructed. By transforming LBA1334 of Agrobacterium tumefaciens, the plant of "complex" was obtained, and the salt-resistant phenotype was analyzed under salt stress, and the transformation of EHA105 from Agrobacterium tumefaciens to Agrobacterium tumefaciens was carried out. The stable transformation lines were obtained, and the salt-resistant phenotypic analysis and physiological index detection were carried out. [results] many bioinformatics software analysis showed that, The fragment contains 1 038 BP ORF and encodes 345 amino acids. GmHAT5 has a theoretical molecular weight of 39.17 KD and a isoelectric point of 4.63 GmHAT5, which are located in the nucleus, just like other HD-Zip family proteins. GmHAT5 contains a homologous domain and a leucine zipper domain. Leucine zipper protein belongs to class I homologous domain. Homologous protein comparison shows that it has the highest homology with wild soybean GsHAT5. The analysis of gene expression characteristics shows that GmHAT5 is expressed in different organs of soybean plant. The results of hairy root transformation showed that after treated with 200 mmol 路L ~ (-1) NaCl for 7 days, the "complex" plants grew well, and the leaves of the control plants were wilted and green. After treated with different concentrations of NaCl for 14 days, the hair roots of the control group were significantly dry and the growth was inhibited compared with the experimental group. The stable transformation results showed that the control group was treated with different concentrations of salt for 14 days. Compared with the control group, the growth status of the GmHAT5 100 vein root was better than that of the two groups, and the relative physiological indexes showed that, compared with the two control groups, the plant growth status was better than that of the control group. The content of malondialdehyde and relative plasma membrane permeability of the transgenic plants decreased significantly, while the chlorophyll content and root activity increased significantly. The results showed that the contents of cations in different plants were determined. Compared with the two groups, the content of Na ~ + in leaves and roots of transgenic Baimai root lines decreased significantly. The contents of K ~ and Ca~(2) in leaves and roots increased significantly. [conclusion] A homologous domain Leucine encoding HD-Zip class I was cloned from soybean. The acid zipper protein gene GmHAT5, which was induced to express in soybean under salt stress, was significantly increased. Overexpression of GmHAT5 significantly enhanced the salt-tolerance ability of the root. Hairy root transformation can be used as a rapid and effective method to screen candidate genes for salt resistance. It is speculated that GmHAT5 plays an important role in the regulation of salt stress in soybean.
【作者单位】：信阳师范学院生命科学学院/大别山农业生物资源保护与利用研究院;
【基金】：国家自然科学基金青年基金(31400213) 信阳师范学院青年骨干教师资助计划(2015);信阳师范学院“南湖学者奖励计划”青年项目 2016年国家级大学生创新创业训练计划(201610477011)
【分类号】：S541.6

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