中国农业科学

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最新录用:玉米转录因子ZmEREB93负调控籽粒发育

庞浩婉1,傅乾坤1,杨青青1,张元元2,付凤玲1,于好强1*   

  1. 1四川农业大学玉米研究所,成都 6111302绵阳师范学院生命科学与技术学院,四川绵阳 621000
  • 出版日期:2022-07-20 发布日期:2022-07-20

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
  • Published:2022-07-20 Online:2022-07-20

摘要: 【目的玉米作为重要的粮、经、饲多用作物,其产量的稳定对经济发展和粮食安全意义重大。AP2/EREBPAPETALA2/ethylene response element binding proteinAP2/EREBP)转录因子在植物生长发育及逆境应答中发挥重要作用。前期研究发现,玉米ZmBES1/BZR1-5转录因子靶基因ZmEREB93可能参与调控种子大小。克隆ZmEREB93并对其表达特性及功能进行分析,为深入解析其调控玉米籽粒发育的功能与机制奠定基础。【方法】从玉米自交系B73中克隆ZmEREB93的全长序列对其基因序列和编码氨基酸序列特征进行生物信息学分析。随后,通过实时荧光定量PCRquantitative real-time PCRqRT-PCR)分析其组织表达模式,分别构建植物和酵母表达载体进行亚细胞定位和转录激活活性分析。经农杆菌介导法将ZmEREB93转入拟南芥,对转基因株系的种子表型进行分析。最后,通过体外染色质免疫共沉淀测序(chromatin immunoprecipitation sequencingChip-seq)和共表达分析筛选ZmEREB93可能调控的候选靶基因,并通过酵母单杂交(yeast one hybrid,Y1H)验证。【结果】成功克隆获得ZmEREB93,序列分析结果表明ZmEREB93无内含子,开放阅读框长618 bp,编码205个氨基酸,有一个高度保守的AP2结构域,属于AP2家族的ERF亚类。qRT-PCR结果表明ZmEREB93在授粉后1525 d的种子中表达量较高,其中25 d种子中表达量最高,约为15 d种子中表达量的11倍,在茎和根中有微量表达,在雄穗、花丝及苞叶中无表达。转录激活试验结果表明,ZmEREB93蛋白在酵母细胞中不具有转录激活活性。亚细胞定位结果显示,ZmEREB93蛋白定位于细胞核。与野生型株系相比,转基因拟南芥株系种子的长和宽显著变小且千粒重显著降低。体外Chip-seq与共表达分析结果表明,Zm00001d013611Zm00001d006016Zm00001d027448Zm00001d039991ZmEREB93转录因子的候选靶基因。Y1H试验表明,ZmEREB93蛋白可直接结合Zm00001d013611启动子。【结论】玉米ZmEREB93作为转录因子在种子中特异性表达,调控种子大小


关键词: 玉米, 转录因子, AP2/EREBP, 籽粒

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-seqand co-expression analysis, and further confirmed by yeast one hybrid (Y1H). 【ResultThe ZmEREB93 gene was cloned by PCR. Sequence analysis showed that ZmEREB93 had no intron and an 618bp ORF, encoding 205 amino acids with a highly conserved AP2 domain and belongs to the ERF subclade of AP2 family. The results of RT-qPCR 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 significant 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. 【ConclusionMaize ZmEREB93 TF specifically expressed in seeds and negatively regulated seed size.


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