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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (9): 1351-1358.doi: 10.3724/SP.J.1006.2020.03008

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2020-09-12 Published:2020-04-21
  • Contact: Jun ZHENG E-mail:13849815737@163.com;zhengjun02@caas.cn
  • 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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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

Table 1

Primers used in this study"

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

Fig. 1

Sequence analysis of ZmCIPK24-2 protein"

Fig. 2

Analysis of the expression pattern of ZmCIPK24-2 A: relative expression of ZmCIPK24-2 after NaCl treatment for indicated time; B: relative expression levels of ZmCIPK24-2 in different tissues of maize. V1: the first leaf is fully expanded; V7: the seventh leaf is fully expanded; R2: the kernel is completed. Bars with different lowercase are significantly different at the 0.05 probability level."

Fig. 3

Molecular analysis of two complementary transgenic lines COM1 and COM2 A: detection of ZmCIPK24-2 transgenic lines; B: transcriptional level of ZmCIPK24-2."

Fig. 4

Salt tolerance of atsos2 can be improved by ZmCIPK24-2 A, B: survival rate of WT, atsos2, COM1 and COM2 plants under salt stress; C, D: root length of WT, atsos2, COM1, and COM2 under salt stress. Mean values and SE were shown from multiple independent experiment, survival rate: n > 70, root length: n > 5. Bars with different lowercase are significantly different at the 0.05 probability level."

Fig. 5

Subcellular localization of ZmCIPK24-2 protein Yellow indicates the yellow fluorescence of YFP protein under confocal laser scanning microscope; Scale bars = 20 μm."

Fig. 6

Interaction between ZmCIPK24-2 and ZmCBLs A: interaction between ZmCIPK24-2 and ZmCBLs by yeast two-hybrid experiment; B: interaction between ZmCIPK24-2 and ZmCBLs by Firefly LCI assay."

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