Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (19): 3685-3696.doi: 10.3864/j.issn.0578-1752.2022.19.001

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

Maize Transcription Factor ZmEREB93 Negatively Regulates Kernel Development

PANG HaoWan1(),FU QianKun1,YANG QingQing1,ZHANG YuanYuan2,FU FengLing1,YU HaoQiang1()   

  1. 1Maize Research Institute, Sichuan Agricultural University, Chengdu 611130
    2College of Life Science & Biotechnology, Mianyang Tearchers’ College, Mianyang 621000, Sichuan
  • Received:2022-04-18 Accepted:2022-06-16 Online:2022-10-01 Published:2022-10-10
  • Contact: HaoQiang YU E-mail:1421781500@qq.com;yhq1801@sicau.edu.cn

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Abstract:

【Objective】 Maize, a kind of crucial crop, is widely used in food supply, livestock feed, and industry. AP2/EREBP (APETALA2/ethylene response element-binding protein) transcription factor (TF) plays an important role in plant growth, development, and stress response. Previous study showed that ZmEREB93 might regulate seed size as a target gene of ZmBES1/BZR1-5 TF. ZmEREB93 was cloned and used to analyze its expression pattern and function, which lays foundation to clarify the function and mechanism of ZmEREB93 regulating seed size. 【Method】 The full length of ZmEREB93 was cloned from maize inbred line B73 by PCR. The characters of nucleotide and amino acid sequences were analyzed by informatic methods. Subsequently, the tissue expression specificity of ZmEREB93 was analyzed via quantitative real time PCR (qRT-PCR). The expression vector in plant and yeast was constructed and used for subcellular localization and transcription activation assay, respectively. ZmEREB93 was transformed into Arabidopsis mediated by agrobacterium transformation. The seed phenotype of transgenic lines was analyzed. Finally, the potential target genes of ZmEREB93 were screened by chromatin immunoprecipitation sequencing (Chip-seq) and co-expression analysis, and further confirmed by yeast one hybrid (Y1H). 【Result】 The ZmEREB93 gene was cloned by PCR. Sequence analysis showed that ZmEREB93 had no intron and a 618 bp ORF, encoding 205 amino acids with a highly conserved AP2 domain and belongs to the ERF subclade of AP2 family. The results of qRT-PCR showed that the ZmEREB93 gene highly expressed in kernels of 15 and 25 days after pollination (DAP), and slightly expressed in stem and root, but did not express in tassel, silk and bract. The expression level of ZmEREB93 was the highest in 25 DAP kernels reached 11 times of that in 15 DAP kernels. The results of transcriptional activation and subcellular localization assay exhibited that ZmEREB93 protein had no transcriptional activation activity in yeast cells and was localized in the nucleus, respectively. Compared to wild type, the seeds of transgenic lines were significantly smaller and showed lower thousand-seed-weight. Chip-seq and co-expression analysis suggested that the Zm00001d013611, Zm00001d006016, Zm00001d027448 and Zm00001d03991 genes were candidate target genes regulated by ZmEREB93 TF. The result of Y1H showed that ZmEREB93 directly bind to Zm00001d013611 promoter. 【Conclusion】 Maize ZmEREB93 TF specifically expressed in seeds and negatively regulated seed size.

Key words: maize, transcription factors, AP2/EREBP, grain

Fig. 1

Amplification of the ORF of ZmEREB93 gene M: 1000 bp ladder DNA marker; 1 and 2: ORF amplification from cDNA and gDNA, respectively"

Fig. 2

Sequence alignment of ZmEREB93 and its homologous proteins AtERF12: Arabidopsis thaliana, AT1G28360; SbERF12: Sorghum bicolor, SORBI_3009G184300; OsERF12: Oryza sativa, OS05G0497200; SiERF12: Setaria italica, XP_004970252.1; AlERF12: Aeluropus littoralis, QYY53011.1; ZjERF12: Ziziphus jujuba, XP_015890508.1; DzERF12: Durio zibethinus, XP_022777342.1; CiERF12: Carya illinoinensis, XP_042944848.1; CqERF12: Chenopodium quinoa, XP_021740383.1; ItERF12: Ipomoea triloba, XP_031112454.1"

Fig. 3

Expression pattern analysis of the ZmEREB93 gene"

Fig. 4

Transcriptional activation activity analysis of ZmEREB93"

Fig. 5

Subcellular localization of ZmEREB93"

Fig. 6

Identification of transgenic lines a: PCR detection of every line; b: GFP detection of every line; M: 1000 bp ladder DNA marker; L#1 and L#2: Transgenic Arabidopsis lines with ZmEREB93, respectively; WT: Wild type. The same as below"

Fig. 7

Seed phenotype of transgenic lines *：P<0.05;**：P<0.01;***：P<0.0001"

Table 1

Target gene of ZmEREB93"

基因ID
Gene ID		 染色体
Chromosome		 富集倍数
Enrichment fold		 相关系数
Correlation
coefficient		 DRE/CRT元件数
DRE/CRT element No.		 基因注释
GO annotation		 拟南芥中同源基因
Homologous gene in Arabidopsis
Zm00001d013611 5 2.69831 0.7976 3 低质量蛋白质：atherin
Low quality protein: atherin		 ERT2（AT4G20880）[35]
Zm00001d006016 2 3.10428 0.7756 3 锌指A20和AN1结构域的应激相关蛋白
Zinc finger A20 and AN1 domain-containing stress-associated protein		 SAP5（AT3G12630）[36]
Zm00001d027448 1 3.88035 0.7052 1 DUF21结构域蛋白
DUF21 domain-containing protein		 AT1G47330
Zm00001d039991 3 2.71538 0.7853 1 未鉴定的 LOC100278084
Uncharacterized LOC100278084		 AT1G13360

Fig. 8

Y1H assay a: Y1H was used to confirm the binding of ZmEREB93 to Zm00001d013611, Zm00001d006016, Zm00001d027448 and Zm00001d039991 promoter. P016, P488, P991 and P611 indicate Zm00001d013611, Zm00001d006016, Zm00001d027448 and Zm00001d039991 promoter, respectively; b: Y1H was used to confirm the binding of ZmEREB93 to different fragments of Zm00001d013611 promoter. P1, P2 and P3 mean different fragment of Zm00001d013611 promoter. △ represents DRE/CRT core element"

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