中国农业科学 ›› 2019, Vol. 52 ›› Issue (22): 3950-3963.doi: 10.3864/j.issn.0578-1752.2019.22.002

• 分子遗传 • 上一篇    下一篇

谷子NADP-ME的鉴定及其对逆境胁迫的响应

赵晋锋,杜艳伟,王高鸿,李颜方,赵根有,王振华,成凯,王玉文(),余爱丽()   

  1. 山西省农业科学院谷子研究所/特色杂粮种质资源发掘与育种山西省重点实验室,山西长治 046011
  • 收稿日期:2019-07-03 接受日期:2019-09-22 出版日期:2019-11-16 发布日期:2019-11-16
  • 通讯作者: 王玉文,余爱丽
  • 作者简介:赵晋锋,Tel:0355-2204195;E-mail:zhaojfmail@126.com
  • 基金资助:
    国家现代农业产业技术体系(CARS-06-13.5-A23);山西省农科院特色技术攻关项目(YGG17021);山西省农科院农业科技创新研究课题(YCX2019T05);国家农业环境数据中心观测检测任务(ZX03S0410)

Identification NADP-ME Gene of Foxtail Millet and Its Response to Stress

ZHAO JinFeng,DU YanWei,WANG GaoHong,LI YanFang,ZHAO GenYou,WANG ZhenHua,CHENG Kai,WANG YuWen(),YU AiLi()   

  1. Millet Research Institute, Shanxi Academy of Agricultural Sciences/Shanxi Key Laboratory of Genetic Resources and Breeding in Minor Crops, Changzhi 046011, Shanxi
  • Received:2019-07-03 Accepted:2019-09-22 Online:2019-11-16 Published:2019-11-16
  • Contact: YuWen WANG,AiLi YU

摘要:

【目的】鉴定谷子NADP-ME家族成员,研究不同成员对非生物逆境胁迫的响应,为揭示SiNADP-ME在谷子逆境应答信号途径中的作用奠定基础。【方法】 利用生物信息学方法鉴定谷子基因组中的NADP-ME家族成员。采用GSDS2.0、plantCARE、Clustalx、MEGA6.0等软件及网站ExPASy对鉴定成员蛋白和基因序列进行生物信息学分析。采用qRT-PCR方法检测SiNADP-ME在苗期不同逆境、不同生育期干旱胁迫及不同光照强度下的表达情况。【结果】 谷子NADP-ME家族由7个成员组成,它们在谷子的第2、3、5、7染色体上呈不均匀分布。保守功能域分析显示7个基因都含有NADP-ME特征保守功能域。序列比对发现谷子NADP-ME成员之间序列非常保守,相似性较高,7个谷子成员序列一致性为77.30%,而不同物种NADP-ME序列之间相似性为56.52%。序列分析显示SiNADP-ME1SiNADP-ME4SiNADP-ME5SiNADP-ME6序列较长,分别编码576、639、652和636个氨基酸,而SiNADP-ME2SiNADP-ME3SiNADP-ME7序列较短,分别编码213、265和149个氨基酸。基因结构分析显示SiNADP-ME1有2个可变剪切,SiNADP-ME5有3个可变剪切,其他基因无可变剪切。SiNADP-ME1SiNADP-ME2SiNADP-ME3SiNADP-ME7含内含子较少,而SiNADP-ME4SiNADP-ME5SiNADP-ME6含内含子较多。蛋白参数预测显示谷子NADP-ME成员间分子量跨度较大,在161.94—725.43 kD,等电点为5.32—8.05,不稳定指数为23.01—45.01,脂肪系数介于89.19—107.77,平均疏水指数介于-0.218—0.004。亚细胞定位预测显示SiNADP-ME成员主要被定位在叶绿体、线粒体和细胞质中。顺式元件分析显示SiNADP-ME成员启动子区域主要包括激素类应答、逆境应答、光应答以及其他类生长调控相关的顺式元件。聚类分析发现谷子SiNADP-ME基因在单、双子叶植物分离之前就已存在。不同物种同源基因对在进化树中广泛存在揭示它们在进化上可能存在共同祖先,也暗示它们在某些信号通路中可能具有相似的功能。苗期逆境表达分析表明所有谷子SiNADP-ME家族基因表达量在本文应用的4种逆境胁迫下都被明显诱导。SiNADP-ME1在ABA、低温、NaCl处理后被诱导的最高相对表达量分别为对照的460.53、411.50和15.24倍;SiNADP-ME6在ABA、低温、PEG、NaCl处理后被诱导的最高相对表达量分别为对照的211.13、15.21、772.41和643.99倍。进一步分析表明SiNADP-ME1SiNADP-ME6在拔节期、抽穗期和灌浆期干旱胁迫下表达量上调。【结论】 从谷子基因组中鉴定了7个NADP-ME基因家族成员;7个成员间序列非常保守并且都含有NADP-ME基因典型特征结构域;7个谷子NADP-ME家族基因参与了植物非生物逆境应答,特别是SiNADP-ME1SiNADP-ME6可能在ABA、盐、干旱、低温等逆境应答信号途径中起重要作用。

关键词: 谷子, NADP依赖型苹果酸酶, 基因表达, 非生物逆境

Abstract:

【Objective】 The objectives of this study are to identify the NADP-ME family genes (SiNADP-MEs) from foxtail millet (Setaria italica) genome, study the response of different members to abiotic stress, and to lay a theoretical foundation for revealing the role of SiNADP-ME genes in stress signal pathway of foxtail millet. 【Method】 The members of NADP-ME family in the foxtail millet genome were identified by bioinformatics methods. The protein and gene sequence of identified members were analyzed using software such as GSDS2.0, plantCARE, Clustalx, MEGA6.0 and the website ExPASy. Real-time quantitative PCR (qRT-PCR) was used to detect the expression levels of SiNADP-ME genes under different stresses at seedling stage, under drought and different light intensities stress at different growth stages. 【Result】 The NADP-ME family of foxtail millet consists of seven members, which were unevenly distributed on chromosomes 2, 3, 5, and 7 of foxtail millet. Conservative functional domain analysis revealed that all seven genes contain the conserved characteristic domains of NADP-ME. Amino acid sequence alignment revealed that the sequences were very conserved and the similarity was very high among the members. The sequence identity of 7 SiNADP-ME members was 77.30%, while the identity of NADP-MEs in different species was 56.52%. Sequence analysis showed that the sequences of SiNADP-ME1, 4, 5, and 6 were longer, encoding 576, 639, 652, and 636 amino acids residues respectively, while the sequences of SiNADP-ME2, 3, and 7 were shorter, encoding 213, 265 amino acids residues respectively. Gene structure analysis showed that SiNADP-ME1 has two alternative transcript, SiNADP-ME5 has three alternative transcript, and other genes have no alternative transcript. SiNADP-ME1, 2, 3 and 7 contain fewer introns, while SiNADP-ME4, 5 and 6 contain more introns. The prediction of protein parameters showed that the molecular weight span among members is large, ranging from 161.94 to 725.43 kD, the isoelectric point from 5.32 to 8.05, the instability index from 23.01 to 45.01, the aliphatic index from 89.19 to 107.77, and the grand average of hydropathicity from -0.218 to 0.004. Subcellular localization predictions show that SiNADP-ME members are mainly localized in chloroplasts, mitochondrial and cytoplasmic. Cis-elements analysis revealed that hormonal, stress, light, and other growth-related cis-elements are present in the promoter region of the SiNADP-ME members. Cluster analysis revealed that SiNADP-ME genes were present before the isolation of monocotyledonous and dicotyledonous plants. The homologous pairs of different species present in the phylogenetic tree revealed that they may evolve from a common ancestor, suggesting that they may have similar functions in certain signaling pathways. Stress expression analysis of seedling stage showed that the expression levels of all the SiNADP-ME family genes were significantly induced under the four stresses applied in this paper. The highest relative expression levels of SiNADP-ME1 under ABA, low temperature and NaCl treatment were 460.53, 411.50 and 15.24 folds than that of the control respectively, while the highest relative expression levels of SiNADP-ME6 under ABA, low temperature, PEG and NaCl treatment were 211.13, 15.21, 772.41 and 643.99 folds than that of the control respectively. Further analysis showed that the expression levels of SiNADP-ME1 and SiNADP-ME6 were up-regulated under drought stress at jointing, heading and filling stage.【Conclusion】 Seven members of NADP-ME gene family were identified from foxtail millet genome. All SiNADP-ME genes contain the typical characteristic domains of the NADP-ME, and their sequences are very conserved. All of the SiNADP-ME genes are involved in plant abiotic stress response, especially SiNADP-ME1 and SiNADP-ME6 may play an important role in response to abiotic stress.

Key words: foxtail millet, NADP-ME, gene expression, abiotic stress