玉米ZmCaM1基因的克隆和表达分析

doi:10.16035/j.issn.1001-7283.2025.04.003

摘要/Abstract

摘要： 在植物应对环境胁迫的反应中，钙调蛋白（CaM）发挥着重要作用。基于玉米（Zea mays L.）自交系B73的转录组数据克隆了一个在干旱胁迫后根系高水平表达的CaM基因，并对其进行了生物信息学分析。结果表明，ZmCaM1基因的编码序列大小为450 bp，编码149个氨基酸。ZmCaM1蛋白不含信号肽且具有亲水性特征。在EF-hand结构域上，ZmCaM1蛋白与高粱（Sorghum bicolor L.）和水稻（Oryza sativa L.）等植物中的CaM蛋白高度保守，呈现相似的二级结构和三级结构。此外，ZmCaM1启动子序列中发现了多个与激素和非生物胁迫相关的顺式作用元件。根据蛋白互作预测和酵母双杂交结果，ZmCaM1蛋白与DNA修复蛋白RAD4互作。通过对原核表达蛋白诱导条件的优化，GST-ZmCaM1在0.1 mmol/L IPTG条件下的表达效果最佳，并经凝胶亲和层析纯化得到了GST-ZmCaM1蛋白。表达分析结果显示，ZmCaM1基因在玉米出苗期的根和叶中表达水平较高。干旱胁迫后该基因在根和叶中上调表达，涝渍胁迫后该基因在根和叶中下调表达，进一步验证了ZmCaM1基因与玉米生长发育以及非生物胁迫响应之间的联系。

关键词: 玉米, 钙调蛋白, 非生物胁迫, 生物信息学, 表达分析

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

贺新春, 杜何为, 黄敏. 玉米ZmCaM1基因的克隆和表达分析[J]. 作物杂志, 2025 (4): 19–28

He Xinchun, Du Hewei, Huang Min. Cloning and Expression Analysis of Maize ZmCaM1 Gene[J]. Crops, 2025(4): 19–28

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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

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

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