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大豆e1-as基因突变体的创制及生理分析

贺红利1,张雨涵1,杨静2,程云清1,赵杨1,李星诺1,司洪亮3,张兴政1,*,杨向东2,*   

  1. 1吉林师范大学, 吉林四平136000; 2吉林省农业科学院, 吉林长春130000; 3吉林师范大学博达学院, 吉林四平136000
  • 收稿日期:2024-09-24 修回日期:2025-04-25 接受日期:2025-04-25 网络出版日期:2025-05-13
  • 基金资助:
    本研究由国家自然科学基金项目(32101649)和大豆基因编辑与新品种培育创新团队项目(20240601063RC)资助。

Creation and physiological analysis of an e1-as gene mutant in soybean

HE Hong-Li1,ZHANG Yu-Han1,YANG Jing2,CHENG Yun-Qing1,ZHAO Yang1,LI Xing-Nuo1,SI Hong-Liang3,ZHANG Xing-Zheng1,*,YANG Xiang-Dong2,*   

  1. 1 Jilin Normal University, Siping 136000, Jilin, China; 2 Jilin Academy of Agricultural Sciences, Changchun 130000, Jilin, China; 3 Boda College, Jilin Normal University, Siping 136000, Jilin, China
  • Received:2024-09-24 Revised:2025-04-25 Accepted:2025-04-25 Published online:2025-05-13
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (32101649) and the Outstanding Talents Team Project of Department of Science and Technology of Jilin Province (20240601063RC).

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摘要：

大豆是光周期高度敏感植物，E1基因是大豆光周期开花机制中的一个核心基因。本研究通过CRISPR/Cas9基因编辑技术构建E1基因编辑载体，并转化大豆Williams 82。对E1基因的PCR扩增产物进行测序发现，T1代植株中有基因编辑发生，进而培育T2代；通过对T2代e1-as-79突变体基因测序发现，E1基因在靶点1处发生了单碱基插入，导致编码序列提前终止。e1-as-79突变体的T3代植株开花时间比野生型大豆早(10±2) d，新出复叶叶绿素的测定值比野生型大豆低(6±2) μg g?1，完全展开复叶叶绿素含量比野生型大豆低(10±4) μg g?1。突变体植株的花粉萌发率比野生型大豆低8%±1%，突变体植株在0.25、0.50、1.00和2.00 h时的花粉管长度分别为(33.96±5.00)、(74.14±5.00)、(142.86±5.00)和(183.50±5.00) μm，而野生型大豆在同等时间上的花粉管长度分别为(46.08±5.00)、(118.89±5.00)、(228.35±5.00)和(307.72±5.00) μm。根据性状对e1-as-79突变体和野生型进行转录组分析，共发现3615个差异基因，其中FT2a和FT5a的表达上升，推测这2个基因可能导致植株早花。

关键词: 大豆, E1基因, 基因编辑, 突变体, 转录组

Abstract: Glycine max is a photoperiod-sensitive plant, and E1 is a key gene regulating flowering time in soybean. As a core component of the photoperiodic flowering pathway, E1 plays a critical role in controlling the timing of floral transition. In this study, we constructed a CRISPR/Cas9-based genome editing vector targeting the E1 gene and introduced it into the soybean cultivar Williams 82. Gene editing was successfully detected in the E1 gene of T1 generation plants, and T2 generation plants were subsequently developed. Sequencing analysis revealed a single base insertion at target site 1 in the E1 gene, resulting in a premature stop codon and truncated protein. Phenotypic analysis showed that the flowering time of the T3 generation was approximately (10±2) days earlier than that of the wild type. In addition, chlorophyll content in the newly emerged trifoliate leaves of the mutant was (6±2) μg g?1 lower than in the wild type, while in fully expanded leaves, it was (10±4) μg g?1 lower. The pollen germination rate in the mutant was 8%±1% lower compared to the wild type. Furthermore, mutant pollen tube lengths measured at 0.25, 0.50, 1.00, and 2.00 h were (33.96±5.00), (74.14±5.00), (142.86±5.00), (183.50±5.00) μm, respectively, whereas the corresponding values in the wild type were (46.08±5.00), (118.89±5.00), (228.35±5.00), and (307.72±5.00) μm. Transcriptome analysis of the e1-as-79 mutant and the wild type identified 3,615 differentially expressed genes, including key flowering regulators FT2a and FT5a, which likely contribute to the early flowering phenotype observed in the mutant.

Key words: soybean, E1 gene, gene editing, mutant, transcriptome

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