Journal of Nuclear Agricultural Sciences ›› 2019, Vol. 33 ›› Issue (12): 2328-2337.DOI: 10.11869/j.issn.100-8551.2019.12.2328

• Induced Mutations for Plant Breeding·Agricultural Biotechnology • Previous Articles     Next Articles

Cloning, Expression and Subcellular Localization Analysis of the Gene AhMKK4 in Peanut (Arachis hypogaea L.)

WANG Mian1, ZHANG Chaoxin2, CHEN Na1, CHEN Mingna1, YU Shanlin1, CHI Xiaoyuan1, *   

  1. 1. Shandong Peanut Research Institute, Qingdao, Shandong 266100;
    2. Qingdao Appearance and Environmental Sanitation Center, Qingdao, Shandong 266100
  • Received:2018-05-11 Revised:2018-08-10 Online:2019-12-10 Published:2019-10-16

花生AhMKK4基因的克隆、表达分析和亚细胞定位研究

王冕1, 张朝昕2, 陈娜1, 陈明娜1, 禹山林1, 迟晓元1, *   

  1. 1. 山东省花生研究所,山东 青岛 266100;
    2. 青岛市市容环境卫生管理中心,山东 青岛 266100
  • 通讯作者: 迟晓元,女,研究员,主要从事花生遗传育种研究。E-mail:chi000@126.com
  • 作者简介:王冕,女,助理研究员,主要从事花生抗逆分子机制研究。E-mail:wmshmilyzcx@126.com
  • 基金资助:
    2014年国家“万人计划”青年拔尖人才(W02070268),国家花生产业技术体系项目(CARS-13),国家自然科学基金项目(31701464),山东省农业科学院青年科研基金项目(2016YQN14)

Abstract: To detect peanut genes associated with stress resistance. A novel mitogen-activated protein kinase kinase gene, named AhMKK4, was isolated from the leaf of peanut (Arachis hypogaea L. cultivar Huayu 20) using PACR-PCR. The full-length cDNA of AhMKK4 is 1 434 bp, including a 966 bp ORF, a 317 bp 5'UTR and a 151 bp 3' UTR. The ORF encodes 322 amino acid protein with the predicted molecular weight of 36.74 kDa, which belongs to the group D MAPKKs in plants. Further, subcellular localization analysis showed that AhMKK4 was located in the nucleus and cytoplasm in plant cells. The analysis of RT-qPCR showed that the highest expression of AhMKK4 was in roots, revealing that AhMKK4 was tissue specific expression gene. The expressions of AhMKK4 were induced by JA and IAA, and were not induced by SA and ABA. These results suggested that AhMKK4 may be involved in JA and IAA mediated signal pathway of peanut. The expression of AhMKK4 was induced distinctly under salt stress, suggesting that AhMKK4 may participate in the salt stress regulation of peanut. This will provide genetic resource for peanut resistance breeding.

Key words: AhMKK4, gene cloning, expression pattern, subcellular localization

摘要: 为挖掘花生抗逆相关基因,本研究以花生品种花育20号为试验材料,根据cDNA文库中已知的促丝裂原活化蛋白激酶激酶(MKK)基因EST序列设计引物,通过RACE-PCR克隆得到AhMKK4基因。结果表明,AhMKK4基因序列全长1 434 bp,含有3'非编码区151 bp,5'非编码区317 bp,开放阅读框全长966 bp,编码一条含有322个氨基酸的蛋白序列。预测其分子量为36.74 kDa,属于MAPKK基因家族D组成员。亚细胞定位显示AhMKK4基因定位于细胞质和细胞核中。RT-qPCR分析发现,AhMKK4基因在根中表达量高于其他组织,说明该基因具有组织表达特异性;AhMKK4基因受JA和IAA诱导时表达量上调,受SA和ABA诱导时表达量下调,说明该基因可能参与到JA和IAA介导的信号转导途径;AhMKK4在盐胁迫下表达量上调,说明该基因可能参与花生对盐胁迫的适应性调控。本研究结果为花生抗逆育种研究提供了新的基因资源。

关键词: 花生促丝裂原活化蛋白激酶激酶4(MKK4), 基因克隆, 表达分析, 亚细胞定位