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作物学报 ›› 2020, Vol. 46 ›› Issue (9): 1351-1358.doi: 10.3724/SP.J.1006.2020.03008

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米ZmCIPK24-2基因在盐胁迫应答中的功能研究

李健1,2(), 王逸茹2, 张凌霄3, 孙明昊1,2, 秦阳2, 郑军1,2,*()   

  1. 1 吉林农业大学农学院, 吉林长春 130118
    2 中国农业科学院作物科学研究所, 北京 100081
    3 北京农学院植物科学技术学院, 北京 102206
  • 收稿日期:2020-02-06 接受日期:2020-03-24 出版日期:2020-09-12 网络出版日期:2020-04-21
  • 通讯作者: 郑军
  • 作者简介:E-mail: 13849815737@163.com
  • 基金资助:
    本研究由国家重点研发计划项目(2016YFD0101002);中国农业科学院创新工程专项经费资助

Functional analysis of ZmCIPK24-2 gene from maize in response to salt stress

LI Jian1,2(), WANG Yi-Ru2, ZHANG Ling-Xiao3, SUN Ming-Hao1,2, QIN Yang2, ZHENG Jun1,2,*()   

  1. 1 College of Agronomy, Jilin Agricultural University, Changchun 130118, Jilin, China
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3 College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China
  • Received:2020-02-06 Accepted:2020-03-24 Published:2020-09-12 Published online:2020-04-21
  • Contact: Jun ZHENG
  • Supported by:
    National Key Research and Development Program of China(2016YFD0101002);Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences

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摘要：

土壤盐化会影响作物正常生长发育, 导致农作物减产。植物在长期适应环境的过程中, 进化出了相应的耐盐分子机制。钙调磷酸酶B类蛋白(CBL)及CBL互作蛋白激酶(CIPK)参与植物对盐胁迫的响应。本研究鉴定到一个拟南芥AtSOS2的同源基因ZmCIPK24-2, 实时荧光定量聚合酶链式反应结果表明ZmCIPK24-2基因在玉米各组织部位广泛表达, 其中在花粉表达量最高; ZmCIPK24-2受盐胁迫诱导表达。ZmCIPK24-2能部分互补拟南芥atsos2突变体的盐敏感表型, 在高盐浓度下转基因株系比atsos2突变体的存活率显著提高, 根长显著增长。亚细胞定位实验表明ZmCIPK24-2定位于细胞质、细胞膜与核膜。利用酵母双杂交实验及LUC互补成像实验发现ZmCIPK24-2与玉米CBLs家族中的ZmCBL1、ZmCBL4、ZmCBL8和ZmCBL9互作。本研究为解析玉米CBL-CIPK信号通路的功能提供了新的实验证据。

关键词: CIPK, CBL, 耐盐性, 玉米

Abstract:

Soil salinity affects the normal growth and development of crops, resulting in reduced crop yields. In the process of long-term adaptation, plants have evolved targeted salt-resistant molecular mechanisms. The calcineurin B-like protein (CBL) and CBL-interaction protein kinase (CIPK) are involved in plant response to salt stress. In this study, we identified an AtSOS2 homologous gene ZmCIPK24-2. The results of real time-qPCR showed that ZmCIPK24-2 gene was ubiquitously expressed in maize, especially in pollen, and was induced by salt stress. It was found that ZmCIPK24-2 could partially complement the salt-sensitive phenotype of the atsos2 mutant. The survival rate and the root length of ZmCIPK24-2-overexpressed lines were significantly increased compared with atsos2 mutant under high salt concentration. Subcellular localization experiments showed that ZmCIPK24-2 was localized in cytoplasm, cell membrane, and nuclear membrane. Yeast two-hybrid experiments and LUC complementation imaging assays showed that ZmCIPK24-2 was interacted with ZmCBL1, ZmCBL4, ZmCBL8, and ZmCBL9, respectively. This study provides a new experimental evidence for the functional analysis of CBL-CIPK signaling pathway in maize.

Key words: CIPK, CBL, salt tolerance, maize

表1

本研究所用的引物"

引物
Primer		 正向序列
Forward sequence (5'-3')		 反向序列
Reverse sequence (5'-3')
ZmCIPK24-2-F/R ATGGCGGGCGCGGGCGCGGG CTAGCAGGTGGTCGTCCTCA
ZmCIPK24-2-Q-F3/R3 AAGGTCCAGCGTCA GGCGTAGATTTGGCA
GAPDH-F/R AGGATATCAAGAAAGCTATTAAGGC GTAGCCCCACTCGTTGTCG
ZmCIPK24-2-RT-F/R TGCCACAACAAAGGAGTTTATCATA TGAGAGATCCAAACCTTGAGATAGT
AtACTIN-F/R GCCAATCCGGTGCTGGTAACA CATACCAGATCCAGTTCCTCCTCCC
ZmCIPK24-2-IF-F/R AGCAGGCTTTGACTTTATGGCGGGCGCGGGCGCGGG TGGGTCTAGAGACTTTCTGCAGGTGGTCGTCCTCA
CBL1-nluc-F/R CACGGGGGACGAGCTCGGTACCATGGGGTGCTTCCATTCCAC ACGCGTACGAGATCTGGTCGACCGTGACGAGATCGTCGA
CBL4-nluc-F/R CACGGGGGACGAGCTCGGTACCATGGGCTGCGCGACGTCCAA ACGCGTACGAGATCTGGTCGACGTCACTGGCTTCTGAAC
CBL8-nluc-F/R CACGGGGGACGAGCTCGGTACCATGGGGTGTGTGTCCTCCAA ACGCGTACGAGATCTGGTCGACCAACTCGTCGTCACTGG
CBL9-nluc-F/R CACGGGGGACGAGCTCGGTACCATGGGGTGCTTCCATTCCAC ACGCGTACGAGATCTGGTCGACCGTGACGAGATCGTCGA
CIPK24-2-cluc-F/R TACGCGTCCCGGGGCGGTACCATGGCGGGCGCGGGCGCGGG TCCTTGTAGTCCATTTGTTGGCAGGTGGTCGTCCTCA

图1

ZmCIPK24-2蛋白的序列分析"

图2

ZmCIPK24-2基因的表达模式分析 A: NaCl处理后ZmCIPK24-2基因的相对表达量; B: ZmCIPK24-2基因在玉米不同组织器官中的相对表达量。Husk leaves: 苞叶; Filament: 花丝; Rachis: 穗轴; Tassel: 雄穗; Pollen: 花粉; Root: 根; Aerial root: 气生根; Leaf: 叶; Stem: 茎; Endosperm: 胚乳。V1: 第1片叶完全展开; V7: 第7片叶完全展开; R2: 籽粒建成。标以不同小写字母的柱值在P<0.05水平差异显著。"

图3

COM1和COM2转基因互补株系的分子检测 A: ZmCIPK24-2转基因株的检测; B: ZmCIPK24-2的转录检测。WT: 野生型; sos2: 突变体。"

图4

ZmCIPK24-2转化拟南芥atsos2突变体能提高atsos2的耐盐性 A, B: 盐胁迫对WT、atsos2、COM1和COM2植株存活率的影响; C, D: 盐胁迫对WT、atsos2、COM1和COM2根长的影响。误差线表示±标准差, 存活率n > 70, 根长n > 5。标以不同小写字母的柱值在P < 0.05水平差异显著。"

图5

ZmCIPK24-2蛋白的亚细胞定位 黄色表示YFP蛋白在激光共聚焦显微镜下所发出的黄色荧光。"

图6

ZmCIPK24-2与ZmCBLs互作 A: 酵母双杂交实验检测ZmCIPK24-2与ZmCBLs的互作; B: LUC互补成像分析ZmCIPK24-2与ZmCBLs之间的相互作用。"

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