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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (6): 1312-1324.doi: 10.3724/SP.J.1006.2022.13040

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

Molecular cloning of two maize (Zea mays) ZmCOP1 genes and their transcription abundances in response to different light treatments

CUI Lian-Hua**(), ZHAN Wei-Min**(), YANG Lu-Hao, WANG Shao-Ci, MA Wen-Qi, JIANG Liang-Liang, ZHANG Yan-Pei*(), YANG Jian-Ping*(), YANG Qing-Hua*()   

  1. College of Agronomy, Henan Agricultural University / State Key Laboratory of Wheat and Maize Crops Science / Center for Crop Genome Engineering, Zhengzhou 450002, Henan, China
  • Received:2021-05-27 Accepted:2021-10-19 Online:2022-06-12 Published:2021-11-02
  • Contact: ZHANG Yan-Pei,YANG Jian-Ping,YANG Qing-Hua E-mail:1970125806@qq.com;630950832@qq.com;zhangyanpei@henau.edu.cn;jpyang@henau.edu.cn;yangqh@henau.edu.cn
  • About author:First author contact:

    ** Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(31871709);Henan Technology Innovation Guidance Project(182106000050);Key Project of Beijing Natural Science Foundation(6151002)

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Abstract:

As an E3 ubiquitin ligase, CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) mediates ubiquitination and degradation of positive regulatory factors such as HY5, LAF1, HFR1, and CO of the light signal transduction in Arabidopsis thaliana, thus mediating seedling photomorphogenesis, anthocyanin synthesis, and flowering time. To explore functional differentiation of ZmCOP1 genes, we cloned two ZmCOP1 genes, designated as ZmCOP1a and ZmCOP1b, from maize inbred line B73 by reverse transcription PCR (RT-PCR). The physicochemical properties, domain prediction and phylogenetic tree of ZmCOP1 were analyzed by bioinformatics software and website. The transcription abundances of two ZmCOP1 genes in different tissues and their responses to different light treatments at seedling stage were further analyzed by quantitative RT-PCR (qRT-PCR). We found that the open reading frames (ORFs) of ZmCOP1a and ZmCOP1b possessed 2082 and 2061 nucleotides, encoding 693 and 686 amino acids, respectively. The COP1 proteins of maize, rice, sorghum, millet, and Arabidopsis shared the same structural domains and high amino acid sequence identity, indicating that they may have similar functions. The two ZmCOP1 genes were mainly expressed in the above-ground tissues. Both of them could rapidly respond to different light treatments, while the transcription abundances of ZmCOP1a were generally higher than those of ZmCOP1b. It might suggest that ZmCOP1a may play more important roles under different light conditions. Under long-day or short-day condition, the transcription abundances of both ZmCOP1a and ZmCOP1b during the dark phase were higher than those during the light phase. Another interesting case is that both genes kept similar expression patterns during the light phase, while, transcription abundances of ZmCOP1b were higher than those of ZmCOP1a during the dark phase. It might imply that ZmCOP1b may be more important than ZmCOP1a in response to long-day or short-day treatment. In conclusion, both ZmCOP1a and ZmCOP1b had got functional redundancy and differentiation. Both genes could participate in different light signal pathways, and regulate maize photomorphogenesis and flowering period. Our results also provide a research foundation for further exploration of two ZmCOP1 genes and application in molecular breeding in maize.

Key words: Zea mays, ZmCOP1, light treatment, gene cloning, transcription abundance

Table 1

Primers for homologous cloning"

基因名称
Gene name		 基因编号
Accession number		 正向引物序列
Forward sequence (5°-3°)		 反向引物序列
Reverse sequence (5°-3°)
ZmCOP1a Zm00001d018207 GGGGGACTCTTGACCATGGGCATGGGCGACTCCTCGGTG AAGTTCTTCTCCTTTACTAGTAGGAGCAAGTACAAGAACTTTTATGG
ZmCOP1b Zm00001d052138 GGGGGACTCTTGACCATGGGCATGGGCGACTCCTCGGTG AAGTTCTTCTCCTTTACTAGTAGGAGCAAGTACATGAACTTTTATGG

Table 2

Primers for qRT-PCR"

基因名称
Gene name		 基因编号
Accession number		 正向引物序列
Forward sequence (5°-3°)		 反向引物序列
Reverse sequence (5°-3°)
ZmCOP1a Zm00001d018207 GCTAACAACGTTCACTCGATAC CAATGCACATCTGATGGTTCAT
ZmCOP1b Zm00001d052138 CTGCTGCGGGCACTACCTTA CCCATTTCATTTCCCTGTTG
ZmUBQ1 Zm00001d015327 ACTTCATGCTTTCGTCCTACGCTCCA CTGGGAGGCTGGTAGTTGATTC

Fig. 1

RT-PCR amplification of both ZmCOP1 genes and identification of pCAMBIA1302-ZmCOP1 constructs using Nco I and Spe I A: RT-PCR amplification of ZmCOP1. M: marker III DNA ladder; 1: RT-PCR product of ZmCOP1a; 2: RT-PCR product of ZmCOP1b; B: the identification of pCAMBIA1302-ZmCOP1 by double enzyme digestion. M: DL10000 DNA ladder; 1 and 2 were digestion products of pCAMBIA1302-ZmCOP1a and pCAMBIA1302-ZmCOP1b using Nco I and Spe I, respectively. The arrow represents the fragment of ZmCOP1."

Fig. 2

Phylogenetic analysis of COP1 protein at amino acid level in maize and other plants The amino acid sequences were obtained from the Ensembl Plants website and the phylogenetic analysis was performed by MAFFT and MEGA software. Amino acid sequence alignment was performed by DNAMAN 8 software. ZmCOP1a: Zea mays, Zm00001d018207; ZmCOP1b: Zea mays, Zm00001d052138; SbCOP1: Sorghum bicolor, KXG31183; SvCOP1: Setaria viridis, TKW41800; SiCOP1: Setaria italica, KQL31700; OsCOP1: Oryza sativa, Os02t0771100; BdCOP1: Brachypodium distachyon, KQK01702; TaCOP1b: Triticum aestivum, TraesCS6D02G305800; TaCOP1c: Triticum aestivum, TraesCS6B02G356400; TaCOP1a: Triticum aestivum, TraesCS6A02G326100; GmCOP1a: Glycine max, KRH14801; GmCOP1b: Glycine max, KRH73341; MeCOP1: Manihot esculenta, OAY35060; GrCOP1: Gossypium raimondii, KJB42844; AtCOP1: Arabidopsis thaliana, AT2G32950; BnCOP1a: Brassica napus, CDY52540; BnCOP1b: Brassica napus, CDY12145; BoCOP1: Brassica oleracea, Bo4g045420."

Fig. 3

Sequence alignment of amino acids and function domains of COP1 proteins from Zea mays, Arabidopsis thaliana, Oryza sativa, Sorghum bicolor, and Setaria italic Multiple sequence alignments at amino acid levels and function domains were analyzed by MAFFT software and SMART website (http://smart.embl-heidelberg.de/) and InterPro website (https://www.ebi.ac.uk/interpro/). AtCOP1: Arabidopsis thaliana COP1, AT2G32950; ZmCOP1a: Zea mays COP1a, Zm00001d018207; ZmCOP1b: Zea mays COP1b, Zm00001d052138; OsCOP1: Oryza sativa COP1, Os02t0771100; SbCOP1: Sorghum bicolor COP1, KXG31183; SiCOP1: Setaria italica COP1, KQL31700."

Fig. 4

Verification of qRT-PCR primers of both ZmCOP1 genes and their relative expression levels in different tissues of maize A: melting curve from qRT-PCR of ZmCOP1a gene. B: melting curve from qRT-PCR of ZmCOP1b gene. C: the relative quantitative standard curve from qRT-PCR and primer amplification efficiency of both ZmCOP1a and ZmCOP1b genes. R2: correlation coefficient; E: primer amplification efficiency, E = 10 ^ (-1/slope) - 1. D: different tissues of maize inbred line B73, including root, young ear, stem, bract, stamen, pulvinus, sheath, filaments, and leaf, were harvested for qRT-PCR analysis. The transcription abundance of ZmCOP1a in the root (set as 1) was used as control. The bar chart shows the relative mean value of three independent biological replicates. The error bars represent the standard deviation. *: P < 0.05; **: P < 0.01."

Fig. 5

Relative expression levels of both ZmCOP1 genes under different continuous light conditions Maize inbred line B73 was grown in continuous darkness (Dk), far-red light (FR, 0.3 µmol m-2 s-1), red light (R, 22.3 µmol m-2 s-1), blue light (B, 13.0 µmol m-2 s-1) or white light (WL, 17.0 µmol m-2 s-1) for 13 days. The transcription abundance of ZmCOP1a under dark conditions (set as 1) was used as control. The bar chart shows the relative mean value of three independent biological replicates. The error bars represent the standard deviation. *: P < 0.05; **: P < 0.01."

Fig. 6

Relative expression level of both ZmCOP1a and ZmCOP1b shifted from darkness to different light conditions After growing in darkness for 11 days, maize B73 seedlings were transferred to red (R, 22.3 µmol m-2 s-1), far-red light (FR, 0.3 µmol m-2 s-1), blue (B, 13.0 µmol m-2 s-1), and white (WL, 17.0 µmol m-2 s-1), respectively. Samples were collected at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h for qRT-PCR analysis. The transcription abundance of ZmCOP1a under dark conditions (set as 1) was used as control. The line chart shows the relative mean value of three independent biological replicates. Error bars represent the standard deviation."

Fig. 7

Relative expression levels of ZmCOP1a and ZmCOP1b under photoperiodic treatment Seedlings of maize inbred line B73 were grown in long-day (LD, 16 h light/8 h darkness) or short-day (SD, 8 h light/16 h darkness) for 13 days, then samples were taken every 2 hours for qRT-PCR analysis. The transcription abundance of ZmCOP1b at 12:00 in the long-day was used as the control (set as 1). The line chart shows the relative mean value of three biological replications. Error bars represent the standard deviation."

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