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作物学报 ›› 2024, Vol. 50 ›› Issue (3): 613-622.doi: 10.3724/SP.J.1006.2024.34093

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

过量表达大豆异丙基苹果酸脱氢酶基因GmIPMDH促进植株开花和生长

刘薇(), 王玉斌, 李伟, 张礼凤, 徐冉, 王彩洁, 张彦威   

  1. 山东省农业科学院作物研究所 / 山东省特色作物工程实验室, 山东济南 250100
  • 收稿日期:2023-06-02 接受日期:2023-09-13 出版日期:2024-03-12 网络出版日期:2023-09-28
  • 通讯作者: *张彦威, E-mail: zywei-1987@163.com
  • 作者简介:E-mail: hnaulw@126.com
  • 基金资助:
    山东省自然科学基金项目(ZR2020MC101);山东省自然科学基金项目(ZR2020QC119);国家自然科学基金项目(32301905);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-04-CES12);济南市高层次人才专项(202228094)

Overexpression of soybean isopropyl malate dehydrogenase gene GmIPMDH promotes flowering and growth

LIU Wei(), WANG Yu-Bin, LI Wei, ZHANG Li-Feng, XU Ran, WANG Cai-Jie, ZHANG Yan-Wei   

  1. Crop Research Institute, Shandong Academy of Agricultural Sciences / Shandong Engineering Laboratory of Featured Crops, Jinan 250100, Shandong, China
  • Received:2023-06-02 Accepted:2023-09-13 Published:2024-03-12 Published online:2023-09-28
  • Contact: *E-mail: zywei-1987@163.com
  • Supported by:
    Natural Science Foundation of Shandong Province(ZR2020MC101);Natural Science Foundation of Shandong Province(ZR2020QC119);National Natural Science Foundation of China(32301905);China Agriculture Research System of MOF and MARA(CARS-04-CES12);High Level Talents Found of Jinan(202228094)

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摘要：

异丙基苹果酸合成酶(isopropylmalate synthase, IPMS)和异丙基苹果酸脱氢酶(isopropylmalate dehydrogenase, IPMDH)是亮氨酸生物合成中的重要限速酶, 但二者在植物生长发育中的功能鲜有报道。本研究对拟南芥AtIPMDH2基因在大豆中的同源基因GmIPMDH进行了克隆和分析。该基因编码的氨基酸序列中含有Iso_dh亚家族保守结构域, 且启动子中含有大量的光反应元件及激素应答元件。实时荧光定量PCR显示大豆叶片中GmIPMDH的表达量随着植株的生长发育逐渐升高。对GmIPMDH进行了烟草的异位表达和大豆的过量表达, 表型分析发现GmIPMDH的过量表达显著提前了烟草和大豆的开花时间, 且株高和节数均显著增加。转录组分析显示, GmIPMDH过量表达大豆叶片中的若干开花相关基因及赤霉素合成相关基因的表达量发生变化, 推测GmIPMDH可能通过赤霉素合成通路参与赤霉素介导的植物开花诱导和株型调控。本研究首次阐明了GmIPMDH在开花期调控中的作用, 为今后进一步研究GmIPMDH调控大豆开花和生长发育的分子机制提供了一定的基础。

关键词: 大豆, 异丙基苹果酸脱氢酶, GmIPMDH, 开花期, 株高

Abstract:

Isopropyl malate synthase (IPMS) and isopropyl malate dehydrogenase (IPMDH) are important rate-limiting enzymes in leucine biosynthesis. However, their functions in plant growth and development have rarely been reported. In this study, we cloned and performed sequence analysis of GmIPMDH, a homologous gene of Arabidopsis AtIPMDH2 in soybean. GmIPMDH contained a conserved domain of Iso_dh, and the promoter of GmIPMDH contained a large number of light and hormonal responsive elements. The qRT-PCR showed that the relative expression level of GmIPMDH in soybean leaves gradually increased with the growth and development of plants. We then performed a function analysis of GmIPMDH by ectopic expression in tobacco and overexpression in soybean. Phenotypic analysis revealed that the overexpression of GmIPMDH significantly promoted flowering of tobacco and soybean. Meanwhile, plant height and nodes number were also increased significantly. Transcriptome analysis displayed that the expression of several flowering-related genes and gibberellin synthesis-related genes were changed in soybean GmIPMDH-overexpression plants. Therefore, we speculated that GmIPMDH may be involved in the gibberellin-mediated flowering and plant type architecture regulation. This study elucidates the role of GmIPMDH in the regulation of flowering time and provides a molecular basis for further research on the mechanism of GmIPMDH regulating soybean flowering and plant growth.

Key words: soybean, isopropylmalate dehydrogenase, GmIPMDH, flowering time, plant height

表1

引物序列"

引物名称 引物序列 目的
Primer name Primer sequence (5'-3') Purpose
GmIPMDH-pro-s AAGTCCTTCGGCTAACAT 用于GmIPMDH启动子的克隆
GmIPMDH promoter cloning
GmIPMDH-pro-a TGTCGTTCCTATCCCATT
GmIPMDH-s ATGGCGGCTTGTCTGCAACT 用于GmIPMDH CDS的克隆
GmIPMDH CDS cloning
GmIPMDH-a TCACACTGCAGCAGCACCAG
35S+GmIPMDH-s ATGACGCACAATCCCACT 用于转基因材料的检测
Transgenic plants detection
35S+GmIPMDH-a ATCAACCCCTTCAGCAAC
qGmIPMDH-s TCCATTTGCCACTGTTCT 用于实时荧光定量PCR
qRT-PCR
qGmIPMDH-a GCATCCTACCAGCTTCGTT
qGmActin4-s GTGTCAGCCATACTGTCCCCATTT
qGmActin4-a GTTTCAAGCTCTTGCTCGTAATCA

图1

GmIPMDH序列分析 A: GmIPMDH基因结构; B: GmIPMDH蛋白质保守结构预测; C: GmIPMDH二级结构分析; 蓝色代表α螺旋; 紫色代表无规则卷曲; 红色代表延伸连; 绿色代表β转角; D: GmIPMDH蛋白三级结构预测。"

表2

GmIPMDH启动子元件分析"

元件
Element		 位置
Position (bp)		 序列
Sequence (5'-3')		 功能
Function
3-AF1 binding site -2037 TAAGAGAGGAA 光应答 Light responsive element
TCT-motif +162 TCTTAC 光应答 Part of a light responsive element
AT1-motif +1886, -1103 AATTATTTTTTATT 光应答 Part of a light responsive module
Box 4 +1242, -723, +1133,
-697, +1170, -883		 ATTAAT 光应答
Part of a conserved DNA module involved in light responsiveness
G-Box -1532, +64 CACGTT/TACGTG 光应答
Cis-acting regulatory element involved in light responsiveness
MYC +772, -356, +666, -42 CATTTG MYC结合位点 MYC-binding site
ABRE +1532, +64 ACGTG 脱落酸应答
Cis-acting element involved in the abscisic acid responsiveness
ERE +250, +220, -248, +185 ATTTTAAA/ ATTTCATA 乙烯应答 Ethylene-responsive element
TCA +780 TCATCTTCAT 水杨酸响应 Salicylic acid responsiveness
STRE +1506 AGGGG 胁迫响应 Stress responsiveness
WUN-motif -997 AAATTACT 创伤响应 Wound responsiveness

图2

GmIPMDH的表达量 A: GmIPMDH基因组织特异性表达; B: 营养生长阶段大豆叶片中GmIPMDH的表达模式。"

图3

GmIPMDH过量表达烟草表型 A: GmIPMDH过量表达烟草开花图示; B: GmIPMDH过量表达烟草开花时间统计; C: 萌发120 d后GmIPMDH过量表达烟草株高统计。**表示在0.01概率水平显著差异(t检验)。"

图4

短日照条件下GmIPMDH过量表达大豆株系的开花时间图示和开花时间统计 A: GmIPMDH过量表达大豆开花图示; B: 不同株系GmIPMDH表达水平; C: 过量表达大豆开花时间统计; D: 播种36 d后大豆株高统计; E: 播种36 d后大豆节数统计。**表示在0.01概率水平显著差异(t检验)。"

图5

田间GmIPMDH过量表达大豆株系的开花时间统计 A: GmIPMDH不同株系中的表达水平; B: 田间GmIPMDH过量表达大豆株系的开花时间统计。**表示在0.01概率水平显著差异。"

图6

GmIPMDH过量表达大豆的转录组学分析 A, B: GmIPMDH过量表达大豆(35S::GmIPMDH)和野生型大豆(Wm82)差异基因表达量层次聚类图; C: 差异表达基因数量; D: 差异表达基因韦恩图; E: 14 d时花期相关差异表达基因表达水平; F: 17 d时花期相关差异表达基因表达水平。"

表3

花期相关差异表达基因功能注释"

基因登录号
Gene ID		 同源基因
Homologs		 通路
Pathways		 时间
Days
Glyma.02G185500 AGL18, AT3G57390 光周期、光感知及信号传导
Photoperiodism, light perception and signaling		 14 d
Glyma.07G132400 AS1, AT2G37630 光周期、光感知及信号传导
Photoperiodism, light perception and signaling
Glyma.15G044400 TOE1, AT2G28550 衰老光周期、光感知及信号传导
Aging photoperiodism, light perception and signaling
Glyma.09G261600 LATE MERISTEM IDENTITY2, AT3G61250 花发育及分生组织特性
Flower development and meristem identity
Glyma.02G121500 SEPALLATA1, AT5G15800 花发育及分生组织特性
Flower development and meristem identity
Glyma.20G153700 SEPALLATA 3, AT1G24260 花发育及分生组织特性
Flower development and meristem identity
Glyma.20G144400 TIC, AT3G22380 节律钟途径
Circadian Clock
Glyma.06G173800 REF6, AT3G48430 整体交互途径
General
Glyma.11G256800 MED13, AT1G55325 整体交互途径
General
Glyma.13G027600 UBP12, AT5G06600 整体交互途径
General
Glyma.15G029000 FPA, AT2G43410 整体交互途径
General
Glyma.19G010200 MBD9, AT3G01460 整体交互途径
General
Glyma.04G022100 FD, AT4G35900 光周期、光感知及信号传导
Photoperiodism, light perception, and signaling		 17 d
Glyma.05G239400 FKF1, AT1G68050 光周期、光感知及信号传导
Photoperiodism, light perception, and signaling
Glyma.07G058200 SPA1, AT2G46340 光周期、光感知及信号传导
Photoperiodism, light perception, and signaling
Glyma.08G141000 NF-YB3, AT4G14540 光周期、光感知及信号传导
Photoperiodism, light perception, and signaling
Glyma.10G204400 TEM1, AT1G25560 光周期、光感知及信号传导
Photoperiodism, light perception, and signaling
Glyma.18G174500 LATE, AT5G48890 光周期、光感知及信号传导
Photoperiodism, light perception, and signaling
Glyma.19G023200 CDF2, AT5G39660 光周期、光感知及信号传导
Photoperiodism, light perception, and signaling
Glyma.15G044400 TOE1, AT2G28550 衰老光周期、光感知及信号传导
Aging photoperiodism, light perception, and signaling
Glyma.10G066800 PETAL LOSS, AT5G03680 花发育及分生组织特性
Flower development and meristem identity
Glyma.20G153700 SEPALLATA 3, AT1G24260 花发育及分生组织特性
Flower development and meristem identity
Glyma.02G146900 TIC, AT3G22380 节律钟
Circadian clock
Glyma.04G228300 PRR5, AT5G24470 节律钟
Circadian clock
Glyma.03G206100 UBP12, AT5G06600 整体交互途径
General
Glyma.04G200500 ICE1, AT3G26744 春化途径
Vernalization
Glyma.07G033800 GA3ox1, AT1G15550 激素途径
Hormones
Glyma.13G218200 GA2ox2, AT1G30040 激素途径
Hormones
Glyma.13G259400 GA2ox1, AT1G78440 激素途径
Hormones
Glyma.20G141200 GA2ox8, AT4G21200 激素途径
Hormones
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