Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (1): 10-29.doi: 10.3864/j.issn.0578-1752.2025.01.002

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

Genome-Wide Identification of Soybean LOX Gene Family and the Effect of GmLOX15A1 Gene Allele on 100-Seed Weight

WANG Wei(), WU ChuanLei, HU XiaoYu, LI JiaJia, BAI PengYu, WANG GuoJi, MIAO Long, WANG XiaoBo*()   

  1. College of Agriculture, Anhui Agricultural University, Hefei 230036
  • Received:2024-05-23 Accepted:2024-06-11 Online:2025-01-01 Published:2025-01-07
  • Contact: WANG XiaoBo

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Abstract:

【Objective】Based on whole genome identification and analysis of soybean LOX gene family members, to understand the taxonomic evolutionary relationships of each member, to study the expression specificity of each gene member in different tissues and their response to abiotic stress, which provided a theoretical basis for further research on the molecular characteristics, evolutionary process, and function of the LOX gene family. 【Method】Based on the LOX protein sequences of rice and Arabidopsis species in Ensembl database, BLASTP alignment of homologous LOX protein sequences in soybean whole genome database was performed, and MEGA X software was used to construct a phylogenetic tree; Using website MEME for protein conserved motif analysis; Using online software GSDS 2.0 to analyze gene structure; Using TBtools for chromosome localization drawing; Analyze soybean LOX family replication genes using McscanX; Using the PlantCARE website to predict the promoter elements of soybean LOX gene family; Draw gene expression heatmaps of soybean under different tissues and abiotic stress using TBtools, and develop molecular markers for the excellent allele variant GmLOX15A1-G/A significantly correlated with 100-seed weight.【Result】A total of 43 LOX genes were identified in soybean, unevenly distributed on 13 chromosomes. Collinearity analysis indicates that the GmLOX gene has undergone extensive replication during the evolutionary process. Meanwhile, 39 different types of cis regulatory elements were detected in the LOX gene promoter, indicating that they may be involved in different pathways such as growth and development, light response, stress response, and hormone induction. Expression pattern analysis revealed that the LOX gene has different levels of expression in different tissues of soybean, indicating that members of this family have tissue and spatiotemporal expression specificity. Under drought stress conditions, the GmLOX gene was significantly differentially expressed in soybean roots and leaves (P<0.05). Among them, GmLOX3A3, GmLOX7A1, GmLOX20B1, GmLOX13A1, and GmLOX20A2 were significantly upregulated or downregulated in roots and leaves, suggesting that the GmLOX gene may play an important role in response to stress. At the same time, it was found that GmLOX15A1 is highly expressed in grain tissue and there is an excellent G/A allele variation in the seventh exon of the gene coding region. Molecular markers were developed for this variant site, and the correlation between different haplotypes of GmLOX15A1 and 100-seed weight was analyzed using 1 200 soybean germplasm resources from different ecological regions over a period of 2 years. The results showed that compared to the GmLOX15A1-A genotype, the average 100-seed weight of soybean germplasm carrying the GmLOX15A1-G allele gene increased by 2.33 g (P<0.001). 【Conclusion】A total of 43 members of the LOX family were identified in soybeans, which can be divided into 3 subfamilies. The promoter region of the GmLOX gene contains a large number of cis acting elements that respond to hormones and stress, playing different roles in drought stress response. Among them, GmLOX15A1 is highly expressed in grain tissue and there is an excellent G/A allele variation in the seventh exon of the coding region of this gene. Compared with the GmLOX15A1-A genotype, the average 100-seed weight of soybean germplasm carrying the GmLOX15A1-G allele gene is significantly increased by 2.33 g. This locus can be used as an excellent haplotype for genetic improvement of soybean grain size.

Key words: soybean, LOX lipoxygenase, bioinformatics, expression analysis, stress response, 100-seed weight

Table 1

Primer sequences used for PCR detection and desired fragment size"

基因
Gene		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 目的基因片段大小
Fragment size (bp)
GmLOX15A1 LOX15A1CAPS-F GAACACCTAGAGCCCAACTTA 712
LOX15A1CAPS-R CTTGCTCGGTTATCACTGGT

Table 2

Analysis of the physicochemical characteristics of GmLOX protein in soybean"

编号
No.		 基因ID
Gene ID		 基因
Gene		 染色体
Chr.		 基因位置
Gene location (bp)		 氨基酸
<BOLD>A</BOLD>mino acid (aa)		 分子量
MW (kDa)		 等电点
pI
1 Glyma.03G091000 GmLOX3A1 03 27013300-27020898 866 99.48 9.04
2 Glyma.03G237300 GmLOX3A2 03 43723483-43730370 818 93.14 6.13
3 Glyma.03G264300 GmLOX3A3 03 45704553-45709164 901 102.13 7.96
4 Glyma.04G105500 GmLOX4A1 04 10440510-10445278 236 27.25 6.64
5 Glyma.04G105900 GmLOX4A2 04 10789400-10791837 227 26.20 5.22
6 Glyma.07G006900 GmLOX7A1 07 503752-509348 859 96.33 6.67
7 Glyma.07G007000 GmLOX7A2 07 509819-514700 864 96.82 6.05
8 Glyma.07G034800 GmLOX7A3 07 2763836-2770113 865 96.34 7.01
9 Glyma.07G034900 GmLOX7B1 07 2782231-2787974 868 97.87 6.49
10 Glyma.07G039900 GmLOX7B2 07 3288274-3294751 927 104.34 7.54
11 Glyma.07G196800 GmLOX7B3 07 36509005-36518982 910 103.04 7.33
12 Glyma.08G102900 GmLOX8A1 08 7886865-7892029 921 103.67 8.10
13 Glyma.08G189200 GmLOX8A2 08 15172904-15178499 860 96.74 7.17
14 Glyma.08G189300 GmLOX8A3 08 15186020-15191205 857 98.68 6.55
15 Glyma.08G189400 GmLOX8A4 08 15193038-15201472 846 94.99 7.17
16 Glyma.08G189500 GmLOX8A5 08 15206364-15212242 867 97.76 5.85
17 Glyma.08G189600 GmLOX8B1 08 15235197-15239971 866 97.17 6.41
18 Glyma.08G189700 GmLOX8B2 08 15242887-15243655 130 15.23 5.00
19 Glyma.08G189800 GmLOX8B3 08 15249052-15254212 868 97.14 7.44
20 Glyma.10G153900 GmLOX10A1 10 38898571-38904504 865 98.16 5.75
21 Glyma.11G130200 GmLOX11A1 11 9903923-9913136 906 103.80 7.38
22 Glyma.11G130300 GmLOX11B1 11 9925260-9936297 901 102.63 6.02
23 Glyma.12G054700 GmLOX12A1 12 3949453-3957386 914 104.41 7.64
24 Glyma.13G030300 GmLOX13A1 13 9773782-9780355 918 104.30 6.80
25 Glyma.13G075900 GmLOX13A2 13 17965521-17975820 803 92.46 7.52
26 Glyma.13G239000 GmLOX13A3 13 34926963-34931964 910 103.77 7.02
27 Glyma.13G347500 GmLOX13A4 13 43761727-43766023 866 97.27 6.66
28 Glyma.13G347600 GmLOX13B1 13 43769021-43773290 826 92.81 6.45
29 Glyma.13G347700 GmLOX13B2 13 43773475-43780320 853 96.46 5.98
30 Glyma.13G347800 GmLOX13B3 13 43797692-43803266 822 92.79 7.00
31 Glyma.14G173500 GmLOX14A1 14 42911150-42913226 231 26.75 5.09
32 Glyma.15G026300 GmLOX15A1 15 2123754-2128104 857 96.77 6.55
33 Glyma.15G026400 GmLOX15A2 15 2130531-2134563 856 96.38 6.81
34 Glyma.15G026500 GmLOX15B1 15 2142191-2147489 853 96.64 5.94
35 Glyma.16G008700 GmLOX16A1 16 735622-742337 922 104.01 7.38
36 Glyma.16G082600 GmLOX16A2 16 9227831-9237983 861 99.16 8.81
37 Glyma.16G096200 GmLOX16B1 16 18245957-18246472 160 18.31 7.66
38 Glyma.19G263300 GmLOX19A1 19 50591809-50596423 899 101.46 7.78
39 Glyma.20G053700 GmLOX20A1 20 12341598-12348808 859 97.25 5.88
40 Glyma.20G054000 GmLOX20A2 20 12411805-12423973 895 101.70 6.31
41 Glyma.20G054100 GmLOX20A3 20 12486755-12493110 860 97.88 5.99
42 Glyma.20G144600 GmLOX20B1 20 38316738-38324822 858 97.94 6.68
43 Glyma.20G234400 GmLOX20B2 20 46695256-46696124 153 17.26 5.94

Fig. 1

Phylogenetic tree construction of LOX gene family members in three species : Arabidopsis thaliana; : Glycine max; : Oryza sativa"

Fig. 2

Chromosomal localization analysis of LOX gene family members in soybean"

Fig. 3

Chromosomal relationships of the GmLOX gene in the soybean genome"

Fig. 4

Collinearity analysis of LOX genes between soybean and three representative species The collinear regions in the genomes of soybean and other species are shown by gray lines. The pairing of homologous LOX genes between soybean and other species were highlighted with a red line"

Fig. 5

Structure of GmLOX members A: Phylogenetic tree; B: The distribution of conserved protein motifs in GmLOX; C: The exon intron structure of the LOX gene. The yellow box represents exons, the black line represents introns, and the green box represents the 5′UTR and 3′UTR regions"

Table 3

The sequence of conserved motifs of GmLOX protein"

基序 Motif 宽度 Width 最佳匹配 Best possible match
1 95 WLLAKAYVVVNDSCYHQLVSHWLNTHAVVEPFVIATNRHLSVVHPIYKLLFPHYRDTMNINSLARKSLVNADGIIEKTFLWGRYALEMSAVVYKD
2 50 APHGLRLVIEDYPYAVDGLEIWDAIKTWVHEYVSLYYPTDAAVQQDTELQ
3 53 AWWKEVVEKGHGDLKDKPWWPKMQTRQELIQSCSTIIWIASALHAAVNFGQYP
4 48 SETPAPLVKYREEELKNVRGDGTGERKEWDRIYDYDVYNDLGDPDKGE
5 36 SKSAWMTDEEFAREMIAGVNPCVIRRLQEFPPQSKL
6 43 YDEMTKNPQKAYLRTITPKFQALVDLSVIEILSRHASDEVYLG
7 26 YATRTILFLQDDGTLKPLAIELSLPH
8 36 GWTSDAQIIQAFKRFGNKLAEIEQKLIQRNNDETLR
9 36 EPNLGGLTVEQAIQNKKLFILDHHDYLIPYLRKINA
10 19 WVFTDQALPADLIKRGMAV

Fig. 6

Predicted cis-elements in the promoter region of soybean LOX genes"

Fig. 7

Analysis of GmLOX gene expression in different tissues"

Fig. 8

Analysis of GmLOX gene expression in different tissues under drought stress a: Root; b: Leaf"

Table 4

Differences in 100-seed weight associated with SNP sites of highly expressed LOX homologous genes in soybean seeds"

序号
No.		 染色体
Chr.		 参考位点
Reference allele		 突变位点
Alternative allele		 物理位置
Position (bp)		 最大值
Max (g)		 最小值
Min (g)		 平均差
Ad (g)		 T P
1 10 A G 38899630 15.69 15.14 0.55 0.91 >0.05
2 13 G C 43764053 15.89 14.38 1.51 2.02 <0.05
3 13 G C 43772226 16.39 14.79 1.60 2.16 <0.05
4 15 G A 2126515 15.76 13.79 1.97 3.21 <0.01
5 20 A C 46695808 15.73 15.40 0.33 0.46 >0.05

Fig. 9

Correlation analysis between SNP loci of GmLOX gene and 100-seed weight"

Table 5

Extreme material varieties information sheet"

编号
No.		 极端材料名称
Extreme material name		 来源
Source		 百粒重均值
Average 100-seed weight (g)
1 ZDD05182 中国黄淮Huang-Huai, China 27.45
2 早生75 Zaosheng75 中国黄淮Huang-Huai, China 26.97
3 石庄大粒黄 Shizhuangdalihuang 中国黄淮Huang-Huai, China 26.71
4 早红豆 Zaohongdou 中国南方Southern China 25.93
5 潢川茶色豆 Huangchuanchasedou 中国黄淮Huang-Huai, China 25.80
6 系19 Xi19 中国黄淮Huang-Huai, China 25.51
7 Kirsches Stamm 2008 国外Abroad 9.96
8 崇庆九月黄 Chongqingjiuyuehuang 中国南方Southern China 11.98
9 猴子毛 Houzimao 中国南方Southern China 13.91
10 M044 未知Not available 10.63
11 灰老鼠皮 Huilaoshupi 中国黄淮Huang-Huai, China 11.64
12 麻黄豆 Mahuangdou 中国黄淮Huang-Huai, China 10.38

Fig. 10

Development of G/A molecular marker for excellent variant loci of GmLOX15A1"

Fig. 11

Correlation analysis between GmLOX15A1-A/G genotype and 100-seed weight"

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