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作物学报 ›› 2022, Vol. 48 ›› Issue (4): 812-824.doi: 10.3724/SP.J.1006.2022.14076

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

GmELF3s调控大豆开花时间和生物钟节律的功能分析

徐昕(), 秦超(), 赵涛, 刘斌, 李宏宇*(), 刘军*()   

  1. 中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2021-04-25 接受日期:2021-07-12 出版日期:2022-04-12 网络出版日期:2021-08-11
  • 通讯作者: 李宏宇,刘军
  • 作者简介:徐昕, E-mail: 935816885@qq.com;
    秦超, E-mail: 82101181010@caas.cn第一联系人：**同等贡献
  • 基金资助:
    中国农业科学院科技创新工程-作物生物信息学及应用项目(2060302-2-20);大豆藏粮于技项目资助(CAAS-ZDRW202003)

Function analysis of GmELF3s in regulating soybean flowering time and circadian rhythm

XU Xin(), QIN Chao(), ZHAO Tao, LIU Bin, LI Hong-Yu*(), LIU Jun*()   

  1. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2021-04-25 Accepted:2021-07-12 Published:2022-04-12 Published online:2021-08-11
  • Contact: LI Hong-Yu,LIU Jun
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences(2060302-2-20);Soybean Grain Storage Technology Program(CAAS-ZDRW202003)

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摘要：

大豆是典型的短日照作物, 光周期的敏感性严重影响大豆的开花时间和产量, 制约大豆的种植范围, 但调控大豆光周期和生物钟节律的机制尚不十分清楚。在模式植物拟南芥中, ELF3与ELF4、LUX一起, 形成ELF4-ELF3-LUX (Evening Complex, EC)生物钟晚间复合物, 在生物钟节律和开花时间调控等方面发挥重要作用。本研究通过CRISPR/Cas9基因编辑系统获得大豆Gmelf3a/j、Gmelf3b-1和Gmelf3b-2的突变体材料。通过观察Gmelf3a/j、Gmelf3b-1和Gmelf3b-2各突变体材料在短日照和长日照下的开花时间发现, GmELF3b-1在长日照下对大豆开花时间起调控作用; 通过观察非纯合双突变体的表型发现, GmELF3a/J与GmELF3b-1和GmELF3b-2之间在调控大豆开花时间方面存在功能冗余。通过qRT-PCR对大豆生物钟节律相关基因的表达进行检测发现, GmCAB、GmPRR9a和GmPRR7a的表达模式发生改变, 这表明GmELF3a/J、GmELF3b-1和GmELF3b-2可能是通过GmPRR9a和GmPRR7a对大豆生物钟节律和开花时间进行调控。

关键词: 光周期, 生物钟节律, 开花时间, 晚间复合物, ELF3

Abstract:

Soybean is a typical short-day crop. Photoperiod sensitivity seriously affects flowering time, yield, and planting range of soybean, but the mechanism underlying photoperiod and circadian rhythm regulation is still unclear. In Arabidopsis thaliana, ELF3, together with ELF4 and LUX, forms the ELF4-ELF3-LUX complex (Evening Complex, EC), which plays an important role in circadian rhythm and flowering time regulation. In this study, soybean mutants of Gmelf3a/j, Gmelf3b-1, and Gmelf3b-2 were obtained by CRISPR/Cas9 gene editing system. We found that GmELF3b-1 regulated the flowering time in soybean under long-day conditions by observing flowering phenotypes in these mutants. The phenotypes of heterozygous double mutants revealed that there was functional redundancy among GmELF3a/J, GmELF3b-1, and GmELF3b-2 in regulating flowering time of soybean. Through detecting the expression of circadian related genes in soybean by using qRT-PCR, it was found that the relative expression patterns of GmCAB, GmPRR9a, and GmPRR7a were changed in soybean. In summary, these results suggested that GmELF3a/J, GmELF3b-1, and GmELF3b-2 may regulate the circadian rhythm and flowering time through GmPRR9a and GmPRR7a in soybean.

Key words: photoperiod, circadian clock, flowering time, Evening Complex, ELF3

表1

构建CRISPR/Cas9-ELF3突变体载体所需引物"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')
GmELF3a-1.1-F1 AATGTGCCACCACATGGATTGTGTATCGGACTGCCCATCC GTTTTAGAGCTAGAAATAGCAA
GmELF3a-1.8-F1 AATGTGCCACCACATGGATTGGTGGCTGCTGACAGTGGGA GTTTTAGAGCTAGAAATAGCAA
GmELF3b1-2.5-F1 AATGTGCCACCACATGGATTGAATATCACGATACTCGGAC GTTTTAGAGCTAGAAATAGCAA
GmELF3b2-3.1-F1 AATGTGCCACCACATGGATTGCGTCGCGGATTTCCGGTTA GTTTTAGAGCTAGAAATAGCAA
GmELF3b2-3.9-F1 AATGTGCCACCACATGGATTGACTGGAGGCCCAATACAGA GTTTTAGAGCTAGAAATAGCAA
gRNA-Xba I-R GCTCGGCAACGCGTTCTAGAAAAAAAAGCACCGACTCGGT
U6-Xba I-F GGAAGCTTAGGCCTTCTAGAAAAATAAATGGTAAAATGTC
U6-R CAATCCATGTGGTGGCACAT

图1

CRISPR/Cas9载体图谱和GmELF3s进化分析 A: CRISPR/Cas9载体示意图; 将表1中设计的gRNA取代图谱中guide RNA的位置, 即完成载体构建。B: 大豆GmELF3s与拟南芥、菜豆(Phaseolus vulgaris)、赤豆(Vigna angularis)、绿豆(Vigna radiata)、小麦(Triticum aestivum L.)、玉米(Zea mays L.)和水稻(Oryza sativa L.) ELF3间的进化分析。"

图2

GmELF3a (A)、GmELF3b-1 (B) and GmELF3b-2 (C)基因靶位点序列图 蓝色部分为PAM序列, 红色部分为gRNA编辑序列。"

图3

短日照条件下Gmelf3s突变体的开花时间和植株高度统计分析 A: 各突变体在短日照下的植株表型; B: 统计学分析Gmelf3a-1.1-1、Gmelf3a-1.8-1、Gmelf3a-1.8-2、Gmelf3b1-2.5-1和Gmelf3b2-3.9-1各突变体的开花时间; C: 统计学分析Gmelf3a-1.1-1、Gmelf3a-1.8-1、Gmelf3a-1.8-2、Gmelf3b1-2.5-1和Gmelf3b2-3.9-1各突变体的植株高度。**表示在0.01水平差异显著。"

图4

长日照条件下Gmelf3s突变体的开花时间图示(A)和开花时间统计(B) *和**分别表示在0.05、0.01水平差异显著。"

图5

双突变体突变位点和短日照下开花时间统计 A~B: 杂合双突变体中Gmefl3a、Gmefl3b-1和Gmefl3b-2的突变序列; (A) 前2排序列为GmELF3a序列和突变后的Gmelf3a突变体序列, 后2排为GmELF3b-1序列和突变后的Gmefl3b-1序列; (B) 前2排序列为GmELF3a序列和突变后的Gmelf3a突变序列, 后2排为GmELF3b-2序列和突变后的Gmefl3b-2序列。C~F: 短日照下杂合双突变体开花时间统计; (C) Gmefl3a/b1-1-1双突变体开花时间图示; (D) Gmefl3a/b1-1-1双突变体开花时间统计图; (E) Gmefl3a/b2-2-1双突变体开花时间图示; (F) Gmefl3a/b2-2-1双突变体开花时间统计图。*和**分别表示在0.05、0.01水平差异显著。"

图6

GmELF3a、GmELF3b-1和GmELF3b-2的生物钟节律分析 A和B分别表示GmELF3a在长日照和短日照下的表达模式; C和D分别表示GmELF3b-1在长日照和短日照下的表达模式; E和F分别表示GmELF3b-2在长日照和短日照下的表达模式。"

附图1

GmELF3a、GmELF3b-1和GmELF3b-2在天隆一号和Gmelf3s各突变体中的生物钟节律分析"

附图2

GmELF3s对生物钟节律基因GmTOC1a、GmPRR5、GmELF4、GmLUX1、GmCCA3、GmREV8a的调控"

图7

GmELF3s对GmCAB、GmPRR9a和GmPRR7a生物钟节律基因的调控 A: Gmelf3a、Gmelf3b-1和Gmelf3b-2突变体中GmCAB的表达模式; B: Gmelf3a、Gmelf3b-1和Gmelf3b-2突变体中GmPRR9a的表达模式; C: Gmelf3a、Gmelf3b-1和Gmelf3b-2突变体中GmPRR7a的表达模式。"

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