Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (18): 3522-3532.doi: 10.3864/j.issn.0578-1752.2024.18.002

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

An EIN3/EIL Family Gene, ZmEIL9 Regulates Grain Development in Maize

ZHU JunJie(), ZHANG XinYue, PAN MengYing, ZHANG JingWen, ZHENG Qi, LI YuLing, DONG YongBin()   

  1. College of Agronomy, Henan Agricultural University/Maize Industry Integration Engineering Technology Research Center of Henan Province, Zhengzhou 450046
  • Received:2024-04-05 Accepted:2024-06-04 Online:2024-09-16 Published:2024-09-29
  • Contact: DONG YongBin

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Abstract:

【Objective】 Grain size and weight are the important factors affecting the yield of maize. The EIN3/EIL gene family is a sort of key transcription factors in the ethylene signaling transduction pathway, and the functions and regulatory mechanisms of the EIN3/EIL gene ZmEIL9 were analyzed in maize kernel development to elucidate its molecular mechanisms.【Method】 The expression patterns of ZmEIL9 in maize kernel at different developmental stages were analyzed by bioinformatics and RT-qPCR. The multiple sequence alignment of ZmEIL9 and its homologs from different species was performed, and the phylogenetic trees was constructed based on the neighbor-joining method. The sequence characteristics of ZmEIL9 protein were analyzed, and subcellular localization of ZmEIL9 was performed. The insertion mutants of Mu transposon and CRISPR/Cas9 knockout mutants of ZmEIL9 were screened, and the agronomic traits including grain filling rate, storage substances such as starch granule and protein content were analyzed. 【Result】 According to the members of EIN3/EIL family in maize, phylogenetic trees showed that ZmEIL9 was closely related to ZmEIL1 and SbEIL1. In the transcriptomic database of maize inbred line B73, the expression levels of ZmEIL9 were higher in the grain at early and late developmental stages. However, the expression levels were higher in inbred line N04 at the middle and late developmental stages. ZmEIL9 encoded 644 amino acids in the inbred lines Dan232 and N04, while its homolog in inbred line B73 has 642 amino acids. Subcellular localization analysis indicated that ZmEIL9 was localized in nucleus. The ZmEIL9 mutants with different Mu transposon insertion sites and CRISPR/Cas9 knockout mutants with amino acid frameshift mutations were obtained, respectively. The plant height, grain length, and 100-grain weight of Mu mutants and knockout mutants were significantly lower than those of its wild counterpart. The grain dry weights at different developmental stages were also analyzed, and the grain filling rates of Zmeil9 mutant were lower than those of the wild type. The starch granules of Zmeil9 mutant were significantly smaller and had an irregular shape based on scanning electron microscopy (SEM) observations. The contents of total starch and the concentration of zein protein in the Zmeil9 mutant were significantly lower than those in the control. 【Conclusion】ZmEIL9 plays an important regulatory role in the kernel development of maize.

Key words: maize (Zea mays L.), EIN3/EIL family, ZmEIL9, grain development, biology function

Fig. 1

Phylogenetic tree of homologous genes of EIN3/EIL family in maize and other plants Zm: Zea mays; OS: Orzya sativa; Sb: Sorghum bicolor; At: Arabidopsis thaliana"

Fig. 2

Expression characteristics of the maize EIN3/EIL gene family in the kernel of development A: Relative expression levels of the EIN3/EIL family in the kernel of inbred line B73; B: Relative expression levels of the EIN3/EIL family in the endosperm of inbred line B73; C: Relative expression levels of ZmEIL1 and ZmEIL9 in the kernel of inbred line N04; S: Seed; en: Endosperm"

Fig. 3

ZmEIL9 cloned and its encoded amino acid sequence A: The amplification of ZmEIL9 in inbred lines N04 and Dan232; B: Amino acid alignment of ZmEIL9"

Fig. 4

Subcellular localization of ZmEIL9 in the maize protoplasts"

Fig. 5

The plant and grain phenotype of Zmeil9 Mu mutants A: Two insertion mutant of Mu transposon in the genetic background of inbred line W22; B, C: The plant and grain phenotype of Zmeil9 and separated wild-type; D-F: The plant height, grain length and 100-grain weight of Zmeil9 and separated wild-type. Grain length is the length of 10 grains (cm). The statistical method is the T-test (* P<0.05; ** P<0.01). The same as below"

Fig. 6

The plant and grain phenotype of Zmeil9 edited mutants A: The characteristics of CRISPR/Cas9 edited sequences in cri1 and cri2; B, C: The plant and grain phenotype of Zmeil9 and wild-type; D-F: The plant height, grain length and 100-grain weight of Zmeil9 and wild-type"

Fig. 7

Grain weight and filling rate of Zmeil9 and control at different developmental stages A: Dry weight of 100 grains; B: Grain filling rate"

Fig. 8

Scanning electron microscopy in the slitting seed of Zmeil9 mutant and control A: The middle part of the kernel; B, C: The outer part of the kernel"

Fig. 9

Content of starch and protein in the kernel of Zmeil9 and control A: Total starch content; B: Zein protein concentration; C: PAGE analysis of zein protein; D: PAGE analysis of non-zein protein"

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