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作物学报 ›› 2024, Vol. 50 ›› Issue (12): 2940-2949.doi: 10.3724/SP.J.1006.2024.41027

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

TaEMF2调控小麦抽穗期的功能分析

武丽芬1,2(), 夏川2, 张立超2, 孔秀英2, 陈景堂1,3,*(), 刘旭1,2,*()   

  1. 1河北农业大学农学院 / 国家玉米改良中心河北分中心 / 华北作物改良与调控国家重点实验室, 河北保定 071001
    2中国农业科学院作物科学研究所 / 作物基因资源与育种全国重点实验室, 北京 100081
    3青岛农业大学农学院, 山东青岛 266109
  • 收稿日期:2024-04-15 接受日期:2024-08-15 出版日期:2024-12-12 网络出版日期:2024-09-02
  • 通讯作者: *陈景堂, E-mail: chenjingtang@126.com; 刘旭, E-mail: liuxu03@caas.cn
  • 作者简介:E-mail: wulifen324826@163.com
  • 基金资助:
    国家重点研发计划项目(2022YFF1002902)

Functional analysis of TaEMF2 in regulating wheat heading date

WU Li-Fen1,2(), XIA Chuan2, ZHANG Li-Chao2, KONG Xiu-Ying2, CHEN Jing-Tang1,3,*(), LIU Xu1,2,*()   

  1. 1College of Agronomy, Hebei Agricultural University / Hebei Sub-Center for National Maize Improvement Center / State Key Laboratory of North China Crop Improvement and Regulation, Baoding 071001, Hebei, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / State Key Laboratory of Crop Gene Resources and Breeding, Beijing 100081, China
    3College of Agronomy, Qingdao Agricultural University, Qingdao 266109, Shandong, China
  • Received:2024-04-15 Accepted:2024-08-15 Published:2024-12-12 Published online:2024-09-02
  • Contact: *E-mail: chenjingtang@126.com; E-mail: liuxu03@caas.cn
  • Supported by:
    National Key Research and Development Program of China(2022YFF1002902)

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摘要：

适宜的抽穗期(开花时间)是作物获得高产和稳产的重要育种目标, EMF2是参与成花调控过程中的重要基因之一, 但该基因在小麦中的功能仍不清楚。本研究从普通小麦中克隆了水稻OsEMF2b的直系同源基因TaEMF2, TaEMF2-2D是一个细胞核和细胞质定位蛋白。敲除TaEMF2会推迟小麦的抽穗期, 过表达TaEMF2-2D会使小麦抽穗期提前。利用RT-qPCR检测小麦开花相关基因在TaEMF2转基因株系中的表达, 结果表明,VRN1和VRN3在敲除转基因株系中的表达水平显著下调, 在过表达株系中这2个基因的表达量则显著上调, 表明TaEMF2可能通过调控VRN1和VRN3的表达来影响小麦的抽穗期。

关键词: 小麦, 抽穗期, TaEMF2, CRISPR/Cas9基因编辑, 过表达

Abstract:

A suitable heading date (flowering time) is a crucial breeding goal for achieving high and stable crop yields. EMF2 is an important gene involved in the regulation of heading date, but its function in wheat remains unclear. In this study, TaEMF2-2D, the orthologous gene of the rice heading date regulatory gene OsEMF2b, was cloned from wheat. Subcellular localization assays revealed that TaEMF2-2D is a nucleus and cytoplasm protein. To elucidate the role of TaEMF2 in regulating wheat heading date, we used the CRISPR/Cas9 gene editing system to knockout TaEMF2, which resulted in a delayed heading date in wheat. Conversely, overexpression of TaEMF2-2D caused an early heading date. Further analysis showed that the expression levels of the flowering-related genes VRN1 and VRN3 were significantly reduced in the knockout lines, while their expression levels were significantly increased in the overexpression plants, as determined by RT-qPCR. These results suggest that the TaEMF2 gene influences wheat heading date by regulating the expression levels of VRN1 and VRN3.

Key words: wheat (Triticum aestivum L.), heading date, TaEMF2 gene, CRISPR/Cas9 gene editing, overexpression

表1

本研究所用引物"

引物名称 Primer name 引物序列 Primer sequence (5'-3')
TaEMF2-2D-CDS-F CATCCTTATCCTTATCCTTAGGGCA
TaEMF2-2D-CDS-R GGGAACGACAAGTGCAAAGTC
pWMB110-TaEMF2-CDS-F GTCGACTCTAGAGGATCCCCGGGATGTGCCGTCAACCGTCG
pWMB110-TaEMF2-CDS-R CATGAATTCCGGCTCGAGACTAGTAAACGTCTTCTTCTTGGGTT
TaEMF2-CDS-SEQF TTGTGTTTCAGATTCAGAA
TaEMF2-CDS-SEQR TGCTCGGCAGAATCGGGTGCT
pBUE413-TaEMF2-F aataatggtctcAAGCgTGTCCTGCTCCAGAAATTTgttttagagctagaaatagc
pBUE413-TaEMF2-R attattggtctcTAAAcGACCACATGTGCCGTCAACcgcttcttggtgcc
pBUE413-413-seq-F TTTCCCAGTCACGACGTTGT
pBUE413-413-seq-R ATCTCTAGAGAGGGGCACGA
TaEMF2-G-2A-P1-F2 TACGCGATGCATTTTGACAGAGT
TaEMF2-G-2A-P1-R2 TATTGTGTTACCTTATAATATGAC
TaEMF2-G-2A-P2-F1 GTGGAAGCTTGCTATCCATGTGC
TaEMF2-G-2A-P2-R1 AATGAGAGCTACCTCTTTTTGCGGT
TaEMF2-G-2B-P1-F1 AATTAAGTTAGGCACCCGTACTTT
TaEMF2-G-2B-P1-R1 GGGGTCTCTCTTTTGAACAATCG
TaEMF2-G-2B-P2-R2 CATGATGGCATTGTTTAATGAGATA
TaEMF2-G-2D-P1-F1 GCATGTAACTTTAGTTGTCCTAG
TaEMF2-G-2D-P2-F1 GGATTGATTGGTGATACGATAG
TaEMF2-G-2D-P2-R1 TCACGTAAATGATAGTAAGTAGTC
TaEMF2-G-2A-P1-seq-R CAGAAGGTCTACAATATAATC
TaEMF2-G-2A-P2-seq-F CTCTAATTATCTATCTATCTATCC
TaEMF2-G-2B-P1-seq-F ATCAACCTTATTTATTTTAAAAAT
TaEMF2-G-2B-P2-seq-F TCTTTGTTAGACACGACCATC
TaEMF2-G-2D-P1-seq-R ATAATCCGTTGCCCTTAAATATG
TaEMF2-G-2D-P2-seq-R ATCAAGGGCTCGTTTTTGGATAACA
RT-TaGAPDH-F TTAGACTTGCGAAGCCAGCA
RT-TaGAPDH-R AAATGCCCTTGAGGTTTCCC
RT-TaEMF2-2D-F3 TGGAGGCGCTGGAGCGGCGGCTA
RT-TaEMF2-2D-R3 GCGCTCCCAGCCCAGCGACCCT
RT-VRN1-5A-F CAGCCTCAAACCAGCTCTTCA
RT-VRN1-5A-R CTCTGCCCTCTCGCCTGT
RT-VRN3-7ABD-F GTCGTTCGGGCAGGAG
RT-VRN3-7ABD-R TCGTGCTCGTACTCTTCCA
pAN-580-GFP-TaEMF2-2D-CDS-F AGCCCAGATCACTAGTATGTGCCGTCAACCGTCG
pAN-580-GFP-TaEMF2-2D-CDS-R TGCTCACCATGGATCCAAACGTCTTCTTCTTGGGTT

图1

小麦TaEMF2基因结构图"

图2

EMF2在高等植物中的进化分析 小麦(Triticum aestivum)、野生二粒小麦(Triticum dicoccoides)、硬粒小麦(Triticum turgidum)、粗山羊草(Aegilops tauschii)、乌拉尔图小麦(Triticum urartu)、长穗偃麦草(Thinopyrum elongatum)、大麦(Hordeum vulgare)、二穗短柄草(Brachypodium distachyon)、水稻(Oryza sativa)、玉米(Zea mays)、高粱(Sorghum bicolor)、向日葵(Helianthus annuus)、银杏(Ginkgo biloba)、绿竹(Bamboosa oldhami)、油菜(Brassica napus)、拟南芥(Arabidopsis thaliana)、大豆(Glycine max)和棉花(Gossypium raimondii)的EMF2间的进化分析。六倍体小麦的同源基因TaEMF2用红色三角标出; 二穗短柄草EMF2用红色方块标出; 玉米和高粱EMF2用红色圆形标出。"

图3

TaEMF2蛋白在小麦原生质体中的亚细胞定位 mCherry: 核定位marker; Merged: TaEMF2与mCherry信号融合; 标尺为10 μm。"

图4

TaEMF2的CRISPR/Cas9基因敲除系的基因型和表型 A: TaEMF2的基因结构和靶点的设计。黑色框表示外显子, 红色线表示靶点的位置, 靶点序列在基因结构下方。B: 基因编辑载体结构示意图。C: TaEMF2敲除系的突变位点序列变化。PAM和sgRNA靶位点分别用红字和黑线表示。蓝色字母表示碱基的插入, 黑色虚线表示缺失的序列。D: TaEMF2敲除系的抽穗期表型。红色箭头指示已抽出的麦穗, 标尺为20 cm。E: TaEMF2敲除系的抽穗天数的统计。数值代表平均值±标准方差(n = 3), ** 表示P < 0.01时达到差异显著水平。"

图5

TaEMF2-2D基因过表达株系的表达量与表型分析 A: 表达量检测, n = 3。B: TaEMF2-2D的过表达株系的抽穗期表型, 红色箭头表示已抽出的麦穗, 标尺为20 cm。C: TaEMF2-2D过表达株系抽穗天数的统计, 数值代表平均值±标准方差(n = 3)。** 表示在P < 0.01差异显著。"

图6

VRN1和VRN3基因在TaEMF2转基因小麦中的表达量分析 A, B: VRN1和VRN3分别在TaEMF2敲除系中的表达水平; C, D: VRN1和VRN3分别在TaEMF2过表达系中的表达水平。TaGAPDH为内参基因。数值代表平均值±标准方差(n = 3)。* 表示在P < 0.05时达到差异极显著水平; ** 表示在P < 0.01差异显著。"

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