大豆e1-as基因突变体的创制及生理分析

doi:10.3724/SP.J.1006.2025.44159

作物学报 ›› 2025, Vol. 51 ›› Issue (8): 2228-2239.doi: 10.3724/SP.J.1006.2025.44159

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 接受日期:2025-04-25 出版日期:2025-08-12 网络出版日期:2025-05-13
通讯作者: *张兴政, E-mail: zxz20051986@163.com;杨向东, E-mail: xdyang020918@126.com
作者简介:E-mail: honglihe2002@126.com
基金资助:
国家自然科学基金项目(32101649);吉林省科技创新创业卓越人才(团队)项目(20240601063RC)

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-Zheng^1,*(), YANG Xiang-Dong^2,*()

¹Jilin Normal University, Siping 136000, Jilin, China
²Jilin Academy of Agricultural Sciences, Changchun 130000, Jilin, China
³Boda College, Jilin Normal University, Siping 136000, Jilin, China

Received:2024-09-24 Accepted:2025-04-25 Published:2025-08-12 Published online:2025-05-13
Contact: *E-mail: zxz20051986@163.com;E-mail: xdyang020918@126.com
Supported by:
National Natural Science Foundation of China(32101649);Outstanding Talents Team Project of Department of Science and Technology of Jilin Province(20240601063RC)

摘要/Abstract

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 hours 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 3615 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

贺红利, 张雨涵, 杨静, 程云清, 赵杨, 李星诺, 司洪亮, 张兴政, 杨向东. 大豆e1-as基因突变体的创制及生理分析[J]. 作物学报, 2025, 51(8): 2228-2239.

HE Hong-Li, ZHANG Yu-Han, YANG Jing, CHENG Yun-Qing, ZHAO Yang, LI Xing-Nuo, SI Hong-Liang, ZHANG Xing-Zheng, YANG Xiang-Dong. Creation and physiological analysis of an e1-as gene mutant in soybean[J]. Acta Agronomica Sinica, 2025, 51(8): 2228-2239.

图/表 14

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表3

表4

与开花相关的下调基因"

基因编码 Gene ID	表达量 log₂FC	上调/下调 Up/down	功能描述 Feature description regulation
Glyma.01G219200	-2.320	Down	花发育的正向调节; 花发育的调控 Positive regulation of flower development; regulation of flower development
Glyma.05G239400	-1.370	Down	花发育的正向调节; 花发育的调控 Positive regulation of flower development; regulation of flower development
Glyma.08G046500	-1.156	Down	花发育的调控 Regulation of flower development
Glyma.03G019400	-3.731	Down	花轮组织; 花组织; 花轮形态发生; 花形态发生; 花器官发育 Floral whorl structural organization; flower structural organization; floral whorl morphogenesis; flower morphogenesis; floral organ development
Glyma.07G081300	-7.698	Down	花轮组织; 花组织; 花轮形态发生; 花形态发生; 花器官发育; 花形态发生 Floral whorl structural organization; flower structural organization; floral whorl morphogenesis; flower morphogenesis; floral organ development; flower morphogenesis
Glyma.12G052300	-1.099	Down	花器官发育 Floral organ development
Glyma.06G051600	-1.471	Down	花器官发育 Floral organ development
Glyma.06G188400	-2.546	Down	花粉管生长调控 Regulation of pollen tube growth
Glyma.07G019800	-4.209	Down	花粉管发育; 长日光周期; 开花 Pollen tube development; long-day photoperiodism; flowering
Glyma.01G219200	-2.320	Down	光周期; 开花 Photoperiodism; flowering
Glyma.02G176700	-1.005	Down	长日照周期开花; 光周期; 开花 Long-day photoperiodism flowering; photoperiodism; Flowering
Glyma.02G09920	-1.223	Down	光周期; 开花Photoperiodism; flowering
Glyma.04G027000	-3.841	Down	DNA结合转录因子活性DNA-binding transcription factor activity
Glyma.06G009100	-7.533	Down	DNA结合转录因子活性DNA-binding transcription factor activity
Glyma.11G112800	-1.614	Down	DNA结合转录因子活性DNA-binding transcription factor activity

表4

图10

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引物名称 Primer name	引物序列 Primer sequence (5°-3°)
e1-T1-F	attgCCTTTCTTTCAACATATAAA
e1-T1-R	aaacTTTATATGTTGAAAGAAAGG
e1-T2-F	gtcaCGCTAACCGATAGCGATTT
e1-T2-R	aaacAAATCGCTATCGGTTAGCG

引物名称 Primer name	引物序列 Primer sequence (5°-3°)
Actin	F: TGTGTTGGACTCTGGTGATGGTGT
Actin	R: TCACATCCCTGACGATTTCTCGCT
FT2a	F: TGATGGGGATTCATCGTTTGGT
FT2a	R: AGAGTGTGGGAGATTGCCAATT
FT5a	F: AATCGCCCTAGGGTTACTGTTG
FT5a	R: ATTCTGGGGTGATGACAGTGTC
IM	F: GGACAAGATTTGGAGGGCCTAA
IM	R: GATCCATTTCATCACCACGCTG
Adagio	F: GGTTTAGCCAGTCAAAGTGCTG
Adagio	R: TCAGTATCCATTCCTCCCCAGT
MYB	F: AGCAATGCCACCTCAAATCAAC
MYB	R: GTCCAAGGCCCTTTCTTCAAAC
EPFL2	F: GCAGCCATGGCAACTTTTCTTA
EPFL2	R: TGTATAGGATCCATGCTGACGC
EPFL1	F: GGCAAGAGGTCAAACCAATCAC
EPFL1	R: GTGGAAGGGTTGAAGGAGAGAG

基因编码 Gene ID	表达量 log₂FC	上调/下调Up/down	功能描述 Feature description regulation
Glyma.08G154500	2.072	Up	花粉管发育; 花粉管生长 Pollen tube development; pollen tube growth
Glyma.08G259300	1.983	Up	花粉管发育; 花粉管生长 Pollen tube development; pollen tube growth
Glyma.10G020800	1.470	Up	花粉管发育; 花粉管生长 Pollen tube development; pollen tube growth
Glyma.13G120800	2.425	Up	花粉管发育; 花粉管生长 Pollen tube development; pollen tube growth
Glyma.15G033600	1.455	Up	花粉管发育Pollen tube development
Glyma.18G283500	3.389	Up	花粉管发育Pollen tube development
Glyma.19G207900	3.095	Up	花粉管发育; 花粉管生长 Pollen tube development; pollen tube growth
Glyma.13G218500	1.082	Up	光周期开花; 花发育Photoperiodic flowering; flower development
Glyma.16G150700	1.407	Up	短日照光周期Short-day photoperiodism
Glyma.13G265000	1.346	Up	短日照光周期; 基因表达调控Short-day photoperiodism; regulation of gene expression
Glyma.01G029300	4.026	Up	植物胚珠发育; 子房发育; 花轮发育 Plant ovule development; pant-type ovary development; floral whorl development
Glyma.04G094800	2.843	Up	植物胚珠发育; 子房发育; 花轮发育 Plant ovule development; plant-type ovary development; floral whorl development
Glyma.06G096500	1.521	Up	植物胚珠发育; 子房发育; 花轮发育; 花器官发育 Plant ovule development; plant-type ovary development; floral whorl development; floral organ development
Glyma.16G044100	2.474	Up	光周期; 开花Photoperiodism; flowering
Glyma.06G205800	1.252	Up	花序分生组织同一性的维持Maintenance of inflorescence meristem identity

大豆e1-as基因突变体的创制及生理分析

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

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[1]	王克晶, 李向华. 我国珍稀的大豆属多年生烟豆和短绒野大豆物种遗传资源濒危性评估分析[J]. 作物学报, 2025, 51(8): 2009-2019.
[2]	孟然, 李赵嘉, 冯薇, 陈悦, 刘路平, 杨春燕, 鲁雪林, 王秀萍. 大豆不同生育时期耐盐性综合评价及耐盐种质筛选[J]. 作物学报, 2025, 51(8): 1991-2008.
[3]	胡蒙, 沙丹, 张晟瑞, 谷勇哲, 张世碧, 李静, 孙君明, 邱丽娟, 李斌. 大豆分枝数QTL定位及候选基因筛选[J]. 作物学报, 2025, 51(7): 1747-1756.
[4]	杨晓慧, 晏宣军, 杨文妍, 付俊杰, 杨琴, 谢玉心. 玉米ZmKL1优异等位基因调控籽粒大小的效应评估及分子机制解析[J]. 作物学报, 2025, 51(6): 1501-1513.
[5]	王琼, 邹丹霞, 陈兴运, 张威, 张红梅, 刘晓庆, 贾倩茹, 魏利斌, 崔晓艳, 陈新, 王学军, 陈华涛. 大豆开花时间和成熟期性状全基因组关联分析与候选基因预测[J]. 作物学报, 2025, 51(6): 1558-1568.
[6]	殷丛丛, 李睿琦, 岳霈尧, 李晨, 牛景萍, 赵晋忠, 杜维俊, 岳爱琴. 基于闭合哑铃介导等温扩增可视化检测大豆花叶病毒SC15方法的建立及应用[J]. 作物学报, 2025, 51(5): 1248-1260.
[7]	王晓琳, 刘忠松, 康雷, 杨柳. 甘蓝型油菜角果长度和每角粒数基因定位以及角果皮转录组动态分析[J]. 作物学报, 2025, 51(4): 888-899.
[8]	张金泽, 周庆国, 杨旭, 王倩, 肖莉晶, 金海润, 欧阳青静, 余坤江, 田恩堂. 芥菜型油菜响应菌核病侵染表达特性与高抗性关联基因分析[J]. 作物学报, 2025, 51(3): 621-631.
[9]	孙程明, 周晓婴, 陈锋, 张维, 王晓东, 彭琦, 郭月, 高建芹, 胡茂龙, 付三雄, 张洁夫. 长链非编码RNA (lncRNA)在甘蓝型油菜分枝角度调控中的功能分析与预测[J]. 作物学报, 2025, 51(3): 559-567.
[10]	钱玉平, 宿兵兵, 高吉星, 阮粉花, 李亚伟, 茅林春. 玉米大豆间作对喀斯特区土壤理化性质及微生物碳代谢特征的影响[J]. 作物学报, 2025, 51(1): 273-284.
[11]	丁树启, 程彤, 王弼琨, 于德彬, 饶德民, 孟凡钢, 赵胤凯, 王晓慧, 张伟. 密植对不同年代大豆品种群体光合生产和产量形成的影响[J]. 作物学报, 2025, 51(1): 161-173.
[12]	聂波涛, 刘德泉, 陈健, 崔正果, 侯云龙, 陈亮, 邱红梅, 王跃强. 北方春大豆品种农艺和品质性状分析与综合评价[J]. 作物学报, 2024, 50(9): 2248-2266.
[13]	叶靓, 朱叶琳, 裴琳婧, 张思颖, 左雪倩, 李正真, 刘芳, 谭静. 联合全基因组关联和转录组分析筛选玉米拟轮枝镰孢穗腐病的抗性候选基因[J]. 作物学报, 2024, 50(9): 2279-2296.
[14]	禹海龙, 吴文雪, 裴星旭, 刘晓宇, 邓跟望, 李西臣, 甄士聪, 望俊森, 赵永涛, 许海霞, 程西永, 詹克慧. 小麦茎秆性状的转录组测序及全基因组关联分析[J]. 作物学报, 2024, 50(9): 2187-2206.
[15]	孙现军, 胡正, 姜雪敏, 王世佳, 陈向前, 张惠媛, 张辉, 姜奇彦. 大豆种质资源苗期耐盐性鉴定评价与筛选[J]. 作物学报, 2024, 50(9): 2179-2186.