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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (3): 613-622.doi: 10.3724/SP.J.1006.2024.34093

• CROP GENETICS & BREEDINGZ·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2024-03-12 Published: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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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

Table 1

Primer sequence"

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

Fig. 1

Sequence analysis of GmIPMDH A: the structure of GmIPMDH; B: the conserved domain prediction of GmIPMDH; chromosomal distribution of GmIPMDH and its homologs; C: the secondary structure analysis of GmIPMDH. The blue lines represent alpha helix, purple lines represent random coil, red lines represent extended strand, green lines represent beta turn; D: the tertiary structure prediction of GmIPMDH protein."

Table 2

Cis-elements of GmIPMDH promoter"

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

Fig. 2

Relative expression pattern of GmIPMDH genes A: tissue specific expression of GmIPMDH; B: the relative expression pattern of GmIPMDH genes in leaf during differential vegetative periods."

Fig. 3

Phenotypes of tobacco with ectopic expression of GmIPMDH A: flowering phenotype of tobacco with ectopic expression of GmIPMDH; B: flowering times in tobacco with ectopic expression of GmIPMDH; C: plant height in tobacco with ectopic expression of GmIPMDH at 120 days after germination. ** represents significant difference at the 0.01 probability level (t-test)."

Fig. 4

Flowering phenotype of GmIPMDH under short-day conditions A: flowering phenotype of GmIPMDH overexpression plants; B: the relative expression level of GmIPMDH in transgenic lines; C: flowering times in wild type plants and GmIPMDH overexpression plants; D: plant height in wild type plants and GmIPMDH overexpression plants at 36 days after planting; E: nodes number in wild type plants and GmIPMDH overexpression plants at 36 days after planting. ** represents significant difference at the 0.01 probability level."

Fig. 5

Flowering time statistics of GmIPMDH overexpression lines in the field A: the relative expression level of GmIPMDH in GmIPMDH overexpression lines; B: flowering time statistics of GmIPMDH overexpression lines in the field. ** represents significant difference at the 0.01 probability level."

Fig. 6

Transcriptome analysis of GmIPMDH overexpression soybean A, B: heat map for cluster analysis of the DEGs (differentially expressed genes) in GmIPMDH overexpression plants (35S::GmIPMDH) compared to the wild type plants (Wm82); C: the number of DEGs; D: Venn diagram of DEGs; E: the relative expression level of DEGs related to flowering time at 14 d after planting; F: the relative expression level of DEGs related to flowering time at 17 d after planting."

Table 3

Functional annotation of DEGs related to flowering time"

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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