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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (6): 1420-1434.doi: 10.3724/SP.J.1006.2024.33060

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

Cloning and functional analysis of ZmGRAS13 gene in maize

SHE Meng1,2(), ZHENG Deng-Yu2, KE Zhao2, WU Zhong-Yi2, ZOU Hua-Wen1,*(), ZHANG Zhong-Bao2,*()   

  1. 1College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China
    2Institute of Biotechnology, Beijing Academy of Agriculture and Forestry Sciences / Beijing Key Laboratory of Agricultural Gene Resources and Biotechnology, Beijing 100097, China
  • Received:2023-10-18 Accepted:2024-01-12 Online:2024-06-12 Published:2024-02-19
  • Contact: * E-mail: zouhuawen@yangtzeu.edu.cn;E-mail: zhangzhongbao@baafs.net.cn
  • Supported by:
    Beijing Natural Science Foundation(6222009);Beijing Academy of Agricultural and Forestry Sciences(KJCX20230203);Beijing Academy of Agricultural and Forestry Sciences Biotechnology Sharing Platform in 2023

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Abstract:

GRAS family is a plant-specific transcription factor, which plays an important role in regulating plant growth and development and responding to stresses. Exploring the function of GRAS family genes in maize (Zea mays L.) provides the important genetic resources for the creation of new maize germplasm. In this study, ZmGRAS13 gene (Zm00001eb401210) was cloned, and its basic characteristics, tissue expression characteristics, and the relative expression patterns under stresses were analyzed by bioinformatics and qRT-PCR. Bioinformatics showed that the full-length coding sequence of this gene was 1638 bp, encoding 545 amino acids. ZmGRAS13 protein had no transmembrane structure, and the molecular weight of 60.79 kD, the theoretical isoelectric point of 5.86, and had a conserved domain unique to the GRAS family. The analysis of 2 kb sequence upstream of the gene promoter indicated that the sequence contained cis-acting elements related to stresses, hormone response, and light response. The qRT-PCR analysis showed that ZmGRAS13 gene was expressed in different tissues of maize, and the relative expression level in stem was the highest. At the same time, the gene has different degrees of induced expression under different abiotic stress treatment conditions. The transient expression experiment of maize protoplasts demonstrated that ZmGRAS13 protein was localized in the nucleus. On 1/2 MS solid medium containing different concentrations of NaCl, mannitol, abscisic acid (ABA), jasmonic acid (JA), and salicylic acid (SA), respectively, the root length of ZmGRAS13 transgenic Arabidopsis lines was significantly longer than the control. In the soil, transgenic Arabidopsis lines grew better than the control under high salt and drought treatments, and the green leaf rate was higher than the control. Compared with the wild type, the content of stress resistance physiological index MDA decreased, the chlorophyll content increased, and the activities of POD and CAT increased in the transgenic ZmGRAS13 Arabidopsis thaliana, and the difference was significant difference. In conclusion, ZmGRAS13 gene may be involved in the regulation of maize growth and development, response to stresses and hormone signal transduction pathway. This study provides an important reference for the further analysis of the biological function of ZmGRAS13 in maize.

Key words: maize, ZmGRAS13, transcription factors, osmotic stress, salt stress, heterologous expression

Table 1

Primers used in this study"

引物名称Primer name 引物序列Primer sequence (5'-3')
pZmGRAS13-FP ACCCGGGCTGCAGAATTCATGCCCATAAAATTTGCACTAG (EcoR I)
pZmGRAS13-RP CATGGTACCCTCGAGAAGCTTGCACCAGGCAGAAGATACGAC (Hind III)
pZmGRAS13RT-FP ATAGGTAGCCCTGACAGTTCCT
pZmGRAS13RT-RP TCCAACATGTAAGCTCCCAGAC
pZmGRAS13T-FP AGGCAGGTAATTGTAGCATG
pZmGRAS13T-RP TCTGTCCAACCATATCCAG
pGAPDHRT-FP
pGAPDHRT-RP		 CCCTTCATCACCACGGACTAC
AACCTTCTTGGCACCACCCT
pActin-FP
pActin-RP		 TACGAGATGCCTGATGGTCAGGTCA
TGGAGTTGTACGTGGCCTCATGGAC

Fig. 1

Cloning and bioinformatics analysis of ZmGRAS13 gene (a): cloning of ZmGRAS13 gene (M: DL2000 DNA marker; 1, 2: ZmGRAS13 fragments amplified); (b): transmembrane structure prediction; (c): protein spatial structure prediction; (d): protein hydrophobicity prediction; (e): conserved domain prediction; (f): analysis of cis-acting elements in promoter region."

Fig. 2

Relative expression level of ZmGRAS13 gene in different tissues of maize Different lowercase letters indicate significant difference at the 0.05 probability level."

Fig. 3

Relative expression level of the ZmGRAS13 gene under different stress treatments in maize (a)?(d): the relative expression level of ZmGRAS13 genes after dehydration, high salt, osmotic and cold treatments, respectively. (e): the relative expression level of ZmGRAS13 genes after JA, ABA, GA, SA, and 2,4-D treatments; *: P < 0.05; **: P < 0.01."

Fig. 4

Subcellular localization of ZmGRAS13 in maize protoplasts GFP: green fluorescence channel; DAPI: DAPI channel; Bright: bright field channel; Merged: merge of GFP, DAPI and Bright channel; pYBA1132:EGFP: protoplast with empty vector; pYBA1132:ZmGRAS13:EGFP: protoplasts transferred into the target gene."

Fig. 5

Identification of T3 generation ZmGRAS13 transgenic Arabidopsis thaliana (a): PCR identification of T3 transgenic Arabidopsis thaliana (M: DL2000 DNA marker; P: positive control; N: negative control; W: water control); (b): qPCR identification of T3 transgenic Arabidopsis thaliana (WT: wild-type Arabidopsis; L-1-L-7: T3 transgenic Arabidopsis lines); *: P < 0.05; **: P < 0.01."

Fig. 6

Comparison of root length between wild type and transgenic Arabidopsis thaliana under different NaCl and mannitol concentrations (A): a?d: 0, 0.10, 0.15, and 0.18 mol L-1 NaCl treatment of Arabidopsis thaliana growth; e: average main root length of Arabidopsis thaliana. (B): a-d: 0, 0.15, 0.20, and 0.30 mol L-1 mannitol treatment of Arabidopsis thaliana growth; e: the average main root length of Arabidopsis thaliana. WT: wild-type Arabidopsis; L-3, L-5, L-7: ZmGRAS13 transgenic Arabidopsis lines; *: P < 0.05; **: P < 0.01."

Fig. 7

Comparison of root length between wild type and transgenic Arabidopsis thaliana under different ABA, JA, and SA concentrations (A): a-d: 0, 10, 25, and 50 μmol L-1 ABA treatment of Arabidopsis thaliana growth; e: the average main root length of Arabidopsis thaliana. (B): a-d: 0, 50, 75, and 100 μmol L-1 JA treatment of Arabidopsis thaliana growth; e: the average main root length of Arabidopsis thaliana. (C): a-d: 0, 25, 50, and 75 μmol L-1 SA treatment of Arabidopsis thaliana growth; e: the average main root length of Arabidopsis thaliana. Abbreviations are the same as those given in Fig. 6; *: P < 0.05; **: P < 0.01."

Fig. 8

Phenotypic analysis of transgenic Arabidopsis under high salt treatment (a): Arabidopsis phenotype; (b): Green leaf rate after high salt treatment; (c): MDA content; (d): POD activity; (e): chlorophyll content; (f): CAT activity. Abbreviations are the same as those given in Fig. 6; *: P < 0.05; **: P < 0.01."

Fig. 9

Phenotypic analysis of transgenic Arabidopsis under drought treatment (a): Arabidopsis phenotype; (b): Green leaf rate before and after drought treatment; (c): MDA content; (d): POD activity; (e): chlorophyll content; (f): CAT activity. Abbreviations are the same as those given in Fig. 6; *: P < 0.05; **: P < 0.01."

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