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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (8): 1920-1933.doi: 10.3724/SP.J.1006.2024.33070

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

Functional study on the regulation of root growth and development and stress tolerance by maize transcription factor ZmEREB180

LIU Chen-Ming1,2(), ZHAO Ke-Yong2(), YUE Man-Fang2, ZHAO Yan-Ming1,*(), WU Zhong-Yi2,*(), ZHANG Chun2,*()   

  1. 1College of Agriculture, Qingdao Agricultural University, Qingdao 266109, Shandong, China
    2Institute of Biotechnology, Beijing Academy of Agriculture and Forestry Sciences / Beijing Key Laboratory of Agricultural Gene Resources and Biotechnology, Beijing 100097, China
  • Received:2023-11-24 Accepted:2024-04-01 Online:2024-08-12 Published:2024-04-19
  • Contact: * E-mail: zhangchun@babrc.ac.cn;E-mail: zwu22@126.com;E-mail: zhaoym796@sina.com
  • About author:** Contributed equally to this work
  • Supported by:
    National Natural Science Foundation of China(32001430);National Natural Science Foundation of China(32372053)

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Abstract:

The AP2/ERF (APETALA2/ethylene-responsive factor) family is one of the largest families of transcription factors in plants, and plays an important role in regulating a variety of biological processes such as plant growth and development, respond-ing to adversity stress, and regulating hormone signaling and substance metabolism. The biological functions of AP2/ERF family genes in many plant species have been validated, but fewer studies have been reported in maize (Zea mays L.). In the previous work, there was significant difference in the relative expression level of the ZmEREB180 transcription factor in root system between the identified critical developmental stages of maize at the six-leaf (V6), the twelve-leaf (V12), and tasseling (VT) stages, the tissue expression analysis revealed that this gene was mainly expressed in maize root system and was significantly higher in young roots than in mature roots, and it was hypothesized that this gene might be involved in the regulation of maize root growth and development. In this study, we cloned the ZmEREB180 (Gene ID: 100192457) transcription factor gene, and preliminarily analyzed the relative expression pattern and biological functions of ZmEREB180 by bioinformatics, RT-qPCR, subcellular localization, and stress-resistant phenotype identification of transgenic Arabidopsis (Arabidopsis thaliana L.) lines. This gene contained two exons and the full-length cDNA was 1023 bp, encoding 340 amino acids. The gene had a conserved domain unique to the AP2/ERF family, which expressed most highly in root system of maize; and the gene had different degrees of induced expression under high salt, drought, high nitrogen, and low nitrogen treatment conditions, with a more rapid and higher expression in response to low nitrogen than high nitrogen; root length of ZmEREB180 transgenic Arabidopsis lines were all significantly longer than the wild type (WT) on 1/2 MS medium containing 0.10 mol L-1, 0.15 mol L-1 NaCl, and 0.15 mol L-1, 0.20 mol L-1, 0.30 mol L-1 mannitol (MNT). Under high salt and drought stress conditions in soil environments, transgenic Arabidopsis lines had healthier growth status, higher green leaf percentage, lower malondialdehyde (MDA) content, and higher peroxidase (POD) activity than WT. The transcription factor ZmEREB180 may play a positive and promotional role in regulating the growth and development of maize root system and enhance the tolerance of maize plants under high salt, drought, osmosis, low nitrogen, and other adversity stresses. This study lays a good foundation for further identification of the biological function and molecular mechanism of transcription factor ZmEREB180 in maize.

Key words: maize (Zea mays L.), ZmEREB180, transcription factor, root system, abiotic stress, nitrogen use efficiency

Table 1

Primers used in this study"

引物名称Primer name 引物序列Primer sequence (5'-3')
pZmEREB180 OE-F CTGAAATCACCAGTCGGTACCATGTGCGGAGGCGCCATCC (Kpn I)
pZmEREB180 OE-R gcccttgctcaccatgGTACCTCAGAAAACAGAACCGTCG (Kpn I)
pZmEREB180RT-F CTGACGAGCTGGCGTTC
pZmEREB180RT-R TCAGAAAACAGAACCGTCG
pGAPDHRT-F CCCTTCATCACCACGGACTAC
pGAPDHRT-R AACCTTCTTGGCACCACCCT
pActinRT-F CCGTGAAGCCAGAAGCTACG
pActinRT-R AACTTGTGGCCGTTTACGTCG
pZmEREB180-F CTGACGAGCTGGCGTTC
pZmEREB180-R TCAGAAAACAGAACCGTCG

Fig. 1

Bioinformatics analysis of ZmEREB180 gene and its protein A: protein spatial structure domain analysis; B: protein transmembrane prediction C: protein tertiary structure prediction; D: promoter sequence analysis."

Fig. 2

Relative expression level of ZmEREB180 in different maize tissues Different lowercase letters indicate significant differences at the 0.05 probability level."

Fig. 3

Relative expression level of ZmEREB180 in different abiotic stress A–D: the relative expression level of ZmEREB180 after treatments with osmotic stress, dehydration, low temperature, and high salt; *: P < 0.05; **: P < 0.01."

Fig. 4

Relative expression level of ZmEREB180 in high nitrogen (HN) and low nitrogen (LN) treatments A-B: the relative expression level of ZmEREB180 after high nitrogen (HN) and low nitrogen (LN) treatments; *: P < 0.05; **: P < 0.01."

Fig. 5

Relative expression level of ZmEREB180 under different phytohormone treatments A-E: the relative expression level of ZmEREB180 after treatments with JA, ABA, SA, 2,4-D, and GA; *: P < 0.05; **: P < 0.01. JA: jasmonic acid; ABA: abscisic acid; SA: salicylic acid; 2,4-D: 2,4-dichlorophenoxyacetic acid; GA: gibberellins."

Fig. 6

Subcellular localisation of ZmEREB180 protein in Nicotiana benthamiana leaves GFP: green fluorescent protein map; Merged: overlay diagram; Bright field: bright field map; CK: nicotiana benthamiana leaf transfected with empty vector; ZmEREB180-EGFP: nicotiana benthamiana leaf transfected with the target gene vector; Bar: 20 μm."

Fig. 7

PCR detection and relative expression level of Arabidopsis thaliana (T3) A: PCR assay of transgenic Arabidopsis thaliana plants (T3); B: RT-qPCR detection of Arabidopsis thaliana plants (T3); M: DL2000 marker; N: negative control; W: water control; WT: wild-type Arabidopsis thaliana; L-1-L-6: T3 transgenic Arabidopsis thaliana lines; **: P < 0.01; ***: P < 0.001."

Fig. 8

Phenotypic analysis of WT and transgenic Arabidopsis thaliana lines under gradient NaCl concentrations A-D: growth of transgenic Arabidopsis thaliana plants and WT plants on 1/2 MS medium treated with 0, 0.10, 0.15, and 0.18 mol L- 1 NaCl, respectively. E: mean data on the length of primary roots of plants on 1/2 MS medium; WT: wild type; L-3, L-5, and L-6: ZmEREB180 transgenic Arabidopsis thaliana lines; Bar: 1.5 cm; *: P < 0.05; **: P < 0.01."

Fig. 9

Phenotypic analysis of WT and transgenic Arabidopsis thaliana lines under gradient mannitol concentrations A-D: growth of transgenic Arabidopsis thaliana plants and WT plants on 1/2 MS medium treated with 0, 0.15, 0.20, and 0.30 mol L-1 mannitol, respectively. E: mean data on the length of primary roots of plants on 1/2 MS medium. Abbreviations are the same as those given in Fig. 8; Bar: 1.5 cm; *: P < 0.05; **: P < 0.01."

Fig. 10

Phenotypic analysis of Arabidopsis thaliana lines under drought treatment in soil A: the phenotype of transgenic and WT Arabidopsis thaliana plants; B: the rate of green leaves under drought treatment; C: MDA content; D: POD activity; E: the rate of green leaves under re-water treatment. Abbreviations are the same as those given in Fig. 8; *: P < 0.05; **: P < 0.01."

Fig. 11

Phenotypic analysis of transgenic lines and WT plants of Arabidopsis thaliana under high salt treatment in soil A: phenotype of transgenic and WT Arabidopsis thaliana plants; B: the rate of green leaves under high salt treatment; C: MDA content; D: POD activity. Abbreviations are the same as those given in Fig. 8; *: P < 0.05; **: P < 0.01."

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