玉米转录因子ZmEREB93负调控籽粒发育  

Maize Transcription Factor ZmEREB93 Negatively Regulates Kernel Development

作　　者：庞浩婉 傅乾坤 杨青青 张元元 付凤玲[1] 于好强 PANG HaoWan;FU QianKun;YANG QingQing;ZHANG YuanYuan;FU FengLing;YU HaoQiang(Maize Research Institute,Sichuan Agricultural University,Chengdu 611130;College of Life Science&Biotechnology,Mianyang Tearchers’College,Mianyang 621000,Sichuan)

机构地区：[1]四川农业大学玉米研究所,成都611130 [2]绵阳师范学院生命科学与技术学院,四川绵阳621000

出　　处：《中国农业科学》2022年第19期3685-3696,共12页Scientia Agricultura Sinica

基　　金：成都市科学技术局技术创新研发项目(2021-YF05-02024-SN);四川省科学技术厅国际科技创新合作项目(2022YFH0067)。

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/EREBP(APETALA2/ethylene response element binding protein,AP2/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PCR(quantitative real-time PCR,qRT-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 sequencing,Chip-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15和25 d的种子中表达量较高,其中,在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,Zm00001d013611、Zm00001d006016、Zm00001d027448及Zm00001d039991为ZmEREB93转录因子的候选靶基因。Y1H试验表明,ZmEREB93蛋白可直接结合Zm00001d013611�【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 Zm EREB93 might regulate seed size as a target gene of ZmBES1/BZR1-5 TF. Zm EREB93 was cloned and used to analyze its expression pattern and function, which lays foundation to clarify the function and mechanism of Zm EREB93 regulating seed size. 【Method】The full length of Zm EREB93 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 Zm EREB93 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. Zm EREB93 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 Zm EREB93 gene was cloned by PCR. Sequence analysis showed that Zm EREB93 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 Zm EREB93gene 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 Zm EREB93 was the highest in 25 DAP kernels reached 11 times of that in 15DAP 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 th

关 键 词：玉米 转录因子 AP2/EREBP 籽粒 

分 类 号：S513[农业科学—作物学]