Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (5): 840-850.doi: 10.3864/j.issn.0578-1752.2025.05.002

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

Cloning of the Promoters and Analysis of Expression Patterns of Maturity Genes E1 and E2 in Soybean

LIU LuPing1(), HU XueJie1, QI Jin1, CHEN Qiang1, LIU Zhi1, ZHAO TianTian1, SHI XiaoLei1, LIU BingQiang1, MENG QingMin1, ZHANG MengChen1, HAN TianFu2(), YANG ChunYan1()   

  1. 1 Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences/Hebei Laboratory of Crop Genetis and Breeding/Huang-Huai-Hai Key Laboratory of Biology and Genetic Improvement of Soybean, Ministry of Agriculture and Rural Affairs, Shijiazhuang 050035
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences/Key Laboratory of Soybean Biology (Beijing), Ministry of Agriculture and Rural Affairs, Beijing 100081
  • Received:2024-07-02 Accepted:2024-08-05 Online:2025-03-07 Published:2025-03-07
  • Contact: HAN TianFu, YANG ChunYan

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Abstract:

【Objective】Maturity time is an essential phenotypic measure of ecological adaptability of soybean and an important trait related to its yield formation. The study of promoters and expression patterns of major maturity genes E1 and E2 would provide basis for the study of gene function and molecular regulatory network of maturity time and lay foundation for adaptability improvement and yield increase in soybean.【Method】The promoter sequences of major maturity genes E1 and E2 were analyzed through the promoter cis-element analysis website PlantCARE, and the important regulatory elements were detected. The promoters of E1 and E2 were cloned, the GUS vectors were constructed, and transformation of Arabidopsis was performed to detect GUS activity in different tissues and organs of transgenic plants. Under low light and strong light conditions, the expression levels of E1 and E2 were compared between long day and short day conditions. The expression levels of E1 and E2 were detected in soybean varieties of different maturity groups, which is for the analysis of correlation between expression levels and maturity time of soybean varieties.【Result】Both E1 and E2 promoters contained multiple photoresponsive elements such as AE-box, Box4 and G-box, E1 promoter also contained auxin-response, abolic acid-response elements, and E2 promoter also contained low temperature-response, drought-response elements and meristem expression elements. In GUS activity detection of transgenic Arabidopsis, E1 promoter had strong transcriptional activity in all organs of the plant, and transcriptional activity of E2 promoter in fibrovascular tissues of seedling hypocotyl, leaf and root was relatively strong. Under both low light and strong light conditions, the expression level of E1 was significantly higher in long day than in short day. Under low light conditions, the expression level of E2 was higher in short day than in long day. Under strong light conditions, the expression level of E2 was higher in long day than in short day. With the increase of maturity time of different soybean varieties, expression level of E1 increased gradually, while E2 expression level did not change regularly.【Conclusion】The promoter of E1 gene was a widely expressed promoter, and its expression level was significantly regulated by photoperiod and significantly correlated with the maturity time of soybean varieties. The promoter of E2 was strongly expressed in vascular tissues of various organs, the photoperiodic regulation mode of this gene was different under strong light and low light conditions, and there was no significant correlation between expression level of E2 and maturity time.

Key words: soybean, maturity time, maturity gene E1, maturity gene E2, promoter, expression pattern

Table 1

Primers used for cloning of soybean E1 and E2 promoters and expression analysis"

引物名称Primer name 引物序列Primer sequence (5′-3′) 用途Purpose
proE1-F CTTCAGACTCAGATGGTAGG E1启动子克隆
Cloning of E1 promoter
proE1-R GTTGGAAGAGATGAATAGGGTC
proE2-F CTTAGTACCACACCCTGGAC E2启动子克隆
Cloning of E2 promoter
proE2-R CTATTTACTTTCAAGGTATTG
E1PT-F CTTCAGACTCAGATGGTAGG E1启动子转基因拟南芥检测
Detection of E1 promoter transgenic Arabidopsis
E1PT-R AGTGTGACTCTACGTTCTCC
E2PT-F AAGTGCAAACTTTATGGAGTA E2启动子转基因拟南芥检测
Detection of E2 promoter transgenic Arabidopsis
E2PT-R CCTCCTATGCAGCTTCAATC
qE1-F CCACCATATGCGAAGCCTCTA E1表达量检测
Detection of E1 expression level
qE1-R GGTGCATGGATTTGGTGTCC
qE2-F CCAAAAATTGAGAATGAATACTCGG E2表达量检测
Detection of E2 expression level
qE2-R TAGTCACTTCGAATACCTACTGGTG
qGmActin-F CGGTGGTTCTATCTTGGCATC 大豆荧光定量内参基因
Reference gene of qRT-PCR in soybean
qGmActin-R GTCTTTCGCTTCAATAACCCTA
qGUS-F CGAAGCGAGCAATGTGATGG GUS表达量检测
Detection of GUS expression level
qGUS-R GATCCGCAAGACGCATCAAC

Fig. 1

The cis-regulatory elements of soybean E1 and E2 promoters A: Cis-regulatory elements of E1 promoter; B: Cis-regulatory elements of E2 promoter. The number below the axis represents the number of bases from the start codon"

Fig. 2

Cloning of soybean E1 and E2 promoters and identification of transgenic Arabidopsis A: Cloning of E1 promoter; B: Identification of pE1 transgenic Arabidopsis; C: Cloning of E2 promoter; D: Identification of pE2 transgenic Arabidopsis. M: DNA Marker; pE1: E1 promoter; pE2: E2 promoter. +: Positive control, the promoter vector is used as the amplification template; -: Negative control, DNA of wild type Arabidopsis is used as the amplification template; H2O: Blank control, water is used as a PCR amplification template; 1-5: The transgenic Arabidopsis plants"

Fig. 3

Chemical staining in different tissues of transgenic Arabidopsis WT: Wild type of Arabidopsis; pE1-GUS: pE1-GUS transgenic Arabidopsis; pE2-GUS: pE2-GUS transgenic Arabidopsis. Bar=1 mm"

Fig. 4

Transcriptional activity of E1 and E2 promoters in transgenic Arabidopsis under long day and short day conditions A: Expression levels of GUS driving by E1 promoter; B: Expression levels of GUS driving by E2 promoter. LD: Long day; SD: Short day. The same as below"

Fig. 5

Expression levels of soybean E1 and E2 under long day and short day conditions A-B: Expression levels of E1 under low light (A) and strong light (B) conditions; C-D: Expression levels of E2 under low light (C) and strong light (D) conditions. Line1-Line4: Near-isogenic lines of E1and E2"

Fig. 6

The expression levels of E1 and E2 in varieties of different maturity groups A-J: Soybean varieties of different maturity groups; A: Heihe 9 (MG 00); B: Fengshou 12 (MG 0); C: Changjihuangdou (MG Ⅰ); D: Huangbaozhu (MG Ⅱ); E: Jidou 12 (MG Ⅲ); F: Yuejin 5 (MG Ⅳ); G: Edou 2 (MG Ⅴ); H: Nannong 493-1 (MG Ⅵ); I: Pingguohuangdou (MG Ⅶ); J: Zigongdongdou (MG Ⅷ). MG: Maturity group"

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