Crops ›› 2025, Vol. 41 ›› Issue (4): 19-28.doi: 10.16035/j.issn.1001-7283.2025.04.003

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Cloning and Expression Analysis of Maize ZmCaM1 Gene

He Xinchun(), Du Hewei, Huang Min()   

  1. School of Life Sciences, Yangtze University, Jingzhou 434025, Hubei, China
  • Received:2024-06-20 Revised:2024-07-30 Online:2025-08-15 Published:2025-08-12

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Abstract:

Calmodulin (CaM) plays an important role in the response of plants to environmental stress. A CaM gene with a high expression level in the root system after drought stress was cloned based on the transcriptome data of the maize (Zea mays L.) inbred line B73, and bioinformatics analysis was conducted. The results showed that the enconding sequence of ZmCaM1 is 450 bp, encoding 149 amino acids. The ZmCaM1 protein lacked a signal peptide and exhibited hydrophilic properties. The EF-hand domain of the ZmCaM1 protein was highly conserved among plants, such as sorghum (Sorghum bicolor L.) and rice (Oryza sativa L.), and presented similar secondary and tertiary structures. There were several cis-acting elements related to hormones and abiotic stress in ZmCaM1 promoter. In addition, we predicted that ZmCaM1 protein could bind the promoter of RAD4 according to bioinformatics methods, and confirmed above results by yeast two-hybrid. By optimizing the induction conditions for the GST-ZmCaM1 protein, we found that the best expression was achieved under 0.1 mmol/L IPTG, and the GST-ZmCaM1 protein was purified via gel affinity chromatography. Expression analysis showed that the expression level of ZmCaM1 gene was higher in roots and leaves of maize at emergence stage. After drought stress, ZmCaM1 was up regulated in roots and leaves, and it was down regulated in roots and leaves under waterlogging stress. The association between the ZmCaM1 gene with the growth and development of maize as well as the response to abiotic stress were verified.

Key words: Maize, Calmodulin, Abiotic stress, Bioinformatics, Expression analysis

Table 1

Informations of primers for PCR and qRT-PCR"

引物名称Primer name 基因ID Gene ID 上游引物Forward primer (5′-3′) 下游引物Reverse primer (5′-3′)
ZmCaM1 Zm00001d007194 ATGGCGGACCAGCTCACC TCACTTGGCCATCATGACCTT
qZmCaM1 Zm00001d007194 TCAAATCGCTCCCTGCCTCT GACCCAGTGATCGCATGACA
BK-CaM1
 Zm00001d007194
 AGGCCGAATTCCCGGGGATCCAT
GGCGGACCAACTCACCG		 CCGCTGCAGGTCGACGGATCCTCAC
TTGGCCATCATGACCTT
AD-AP1G1
 Zm00001d045195
 GCCATGGAGGCCAGTGAATTCATGG
ACCTCGCCATCAATCC		 ACGATTCATCTGCAGCTCGAGCTACA
ACCCAGCAGGAAAGTTGC
AD-AP1G2
 Zm00001d036305
 GCCATGGAGGCCAGTGAATTCAT
GGACCTCGCCATCAACC		 ACGATTCATCTGCAGCTCGAGCTACAAC
CCAGAAGGAAAGTTGC
AD-RAD4
 Zm00001d032245
 GCCATGGAGGCCAGTGAATTCA
TGCGGCGGACGAGGAGC		 ACGATTCATCTGCAGCTCGAGTTACAAC
TCCTCTACTTGGATAGAAAAAC
GST-CaM1
 Zm00001d007194
 GATCTGGTTCCGCGTGGATCCATGGCGG
ACCAGCTCACC		 GTCACGATGCGGCCGCTCGAGTCA
CTTGGCCATCATGACCTT
qZmActin Actin TTGCTATCCAGGCTGTTCTT CGACCTCAGCAGCGCGGTCA

Fig.1

CDS amplification of ZmCaM1 gene (a) and the colony PCR amplification (b) Lines 1-6, six different colonies; M: 2000 bp DNA marker."

Table 2

Physicochemical properties of ZmCaM1 protein"

一级结构特征
Characteristics of primary structure		 预测结果
Prediction result
氨基酸数量Number of amino acids 149
分子量Molecular weight (kDa) 16 831.67
分子式Molecular formula C722H1135N189O253S10
带正电荷残基总数
Total number of positively charged residues		 15
带负电荷残基总数
Total number of negatively charged residues		 38
平均疏水性Average hydrophobicity -0.602
脂肪指数Aliphatic index 70.07
不稳定系数Instability coefficient 23.23
半衰期Estimated half-life (h) 30.00
等电点Isoelectric point 4.11

Fig.2

ZmCaM1 protein hydrophilicity/hydrophobicity prediction (a), signal peptide prediction (b), transmembrane structure prediction (c) and phosphorylation sites prediction (d)"

Fig.3

Comparison of ZmCaM1 amino acid sequences with other 20 homologous sequences Blue: Similarity 100%; Pink: Similarity greater than or equal to 75%; Yellow: Similarity greater than or equal to 50%."

Fig.4

Construction of evolutionary tree and conserved domains of ZmCaM1"

Table 3

The conserved motifs of ZmCaM proteins"

Motif编号Motif number Motif序列Motif sequence
Motif 1 GCITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLNLMAR
Motif 2 FRVFDKDQNGFISAAELRHVMTNLGEKLTDEEVDEMIREADVDGDGQINY
Motif 3 MADQLTDDQISEFKEAFSLFD
Motif 4 EEFVKVMMAKRRRKR
Motif 5 KMKDTDSEEEL
Motif 6 PSSSTERKEERRRRKSHCRIL
Motif 7 HNPSQP
Motif 8 GIWKYAASKLLEIRKSHKRFP
Motif 9 IQEKRRGSEPERSPSPQK
Motif 10 LNHTNV

Fig.5

Secondary structure (a) and tertiary structure (b) of ZmCaM1 protein"

Fig.6

Cis-acting elements of ZmCaM1 promoter"

Fig.7

Prediction of ZmCaM1 protein interactions network"

Fig. 8

The interaction validation between ZmCaM1 and other proteins by yeast two-hybrid"

Fig.9

Analysis of prokaryotic expression (a) and purification (b) of ZmCaM1"

Fig.10

The relative expression levels of ZmCaM1 gene in tissues at various stages (a), and under waterlogging (b) and drought stress treatments (c) The different lowercase letters indicate significant difference at P < 0.05 level."

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