大豆油菜素内酯信号响应转录因子GmBEE1和GmBEE3的克隆及其表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0381

摘要/Abstract

摘要：

目的探究大豆油菜素内酯信号响应转录因子GmBEE1a和GmBEE3的生物学功能，为大豆抗病与高产基因资源的挖掘提供科学依据。方法以南豆12为供试材料，采用生物信息学分析GmBEEs基因结构、编码蛋白的理化性质及其空间构象，克隆GmBEE1a和GmBEE3基因编码序列（CDS）片段，并观察蛋白的亚细胞定位；通过实时定量荧光PCR技术分析GmBEEs基因的组织特异性表达及对不同处理的响应特性。结果大豆GmBEE1a和GmBEE3均含有6个外显子和5个内含子，属于典型的bHLH类转录因子，编码酸性不稳定蛋白；系统进化分析揭示GmBEE1a和GmBEE3蛋白与拟南芥、烟草同源蛋白亲缘关系较近；启动子顺式作用元件分析显示，两个基因启动子区域均包含光响应、胁迫应答及激素调控等元件；烟草瞬时表达实验表明，GmBEE1a和GmBEE3蛋白定位在细胞核中。RT-qPCR结果表明，GmBEE1a和GmBEE3在大豆花和叶中的表达水平高于其他组织；根腐病尖孢镰孢菌B3S1和细菌鞭毛蛋白多肽flag22处理均显著诱导2个GmBEEs表达，但根际促生绿针假单胞菌IRHB3处理则抑制其表达；此外，外源水杨酸（SA）、茉莉酸甲酯（MeJA）、油菜素内酯（BL）处理后，GmBEE1a 和 GmBEE3均被显著瞬时诱导表达。结论大豆bHLH转录因子GmBEE1a和GmBEE3不仅参与调控大豆生长发育，而且能响应微生物侵染及外源激素调控。

关键词: 大豆, bHLH 转录因子, GmBEE基因, 表达模式

Abstract:

Objective This study aims to explore the biological functions of soybean transcription factors GmBEE1a and GmBEE3 involving in brassinosteroid signaling, and provides a scientific basis for the discovery of soybean disease resistance and high-yield gene resources. Method The soybean variety Nandou 12 (Glycine max cv. Nandou 12) was used as the material, bioinformatics methods were employed to analyze GmBEEs' gene structure, physicochemical properties of the encoded proteins, and their spatial conformations, the gene coding sequences (CDS) of GmBEE1a and GmBEE3 were cloned, and the subcellular localizations of the proteins were observed using the tobacco transient expression system. Real-time quantitative PCR was used to analyze the tissue-specific expressions of the two GmBEEs genes and their expression characteristics under different treatments. Result Both GmBEE1a and GmBEE3 in soybean contained six exons and five introns, and identified as the typical bHLH transcription factor. Their encoded proteins were acidic and unstable. Phylogenetic analysis revealed that GmBEE1a and GmBEE3 proteins were closely related to homologous proteins in Arabidopsis thaliana and tobacco (Nicotinan tabacum). Promoter cis-acting element analysis showed that the promoter regions of both genes contained functional elements related to light response, stress response, and hormone regulation. Both Bioinformatics predictions and tobacco transient expression experiments indicated that GmBEE1a and GmBEE3 proteins were localized in the nucleus. Tissue-specific expression analysis revealed that the expressions of GmBEE1a and GmBEE3 in soybean flowers and leaves were higher than those in the seeds, roots, stems, and pods. Treatment with the pathogen strain Fusarium oxysporum B3S1 and the bacterial flagellin peptide flg22 significantly induced the expressions of both GmBEEs, whereas treatment with rhizosphere beneficial bacterial strain Pseudomonas chlororaphis IRHB3 suppressed their expression. Additionally, exogenous treatments with salicylic acid (SA), methyl jasmonate (MeJA), and brassinolide (BL) transiently and significantly induced the expressions of GmBEE1a and GmBEE3, followed by a reduction in expression. Conclusion The soybean bHLH transcription factors GmBEE1a and GmBEE3 not only participate in regulating soybean growth and development but also respond to microbial colonization and exogenous hormone treatments.

Key words: soybean, transcription factor bHLH, GmBEE gene, expression pattern

何文馨, 陈玲, 黄灵琳, 徐焱, 孙祖栋, 曾维英, 常小丽, 杨峰. 大豆油菜素内酯信号响应转录因子GmBEE1和GmBEE3的克隆及其表达模式分析[J]. 生物技术通报, 2025, 41(11): 261-271.

HE Wen-xin, CHEN Ling, HUANG Ling-lin, XU Yan, SUN Zu-dong, ZENG Wei-ying, CHANG Xiao-li, YANG Feng. Cloning and Expression Pattern Analysis of Brassinosteriod Signal Response Transcription Factors GmBEE1 and GmBEE3 in Soybean[J]. Biotechnology Bulletin, 2025, 41(11): 261-271.

图/表 11

表 1 用于基因克隆及实时荧光定量PCR扩增的引物序列

Table 1 Primer sequences for gene cloning and real-time fluorescence quantitative PCR amplification

引物名称 Primer name	上游引物 Forward primer （5′‒3′）	下游引物 Reverse primer （5′‒3′）
GmBEE1a-CDS	ATGGCAGAATTCACTGCAGATTT	CAGGGGCCATGACCATGTT
GmBEE3-CDS	ATGGCTGAATTCACTGCAGATTT	CAGAGGCCATGGCCATGTT
qPCR-GmBEE1a	CAGAACATTGTCCCAGGATG	ATTCTTCTCTCACATACCTGTC
qPCR-GmBEE3	CCGGCTCTTGTTCAATGTGTC	TAGCTTGACCACGTTTGGCT
eYFP	GAAGATGCCTCTGCCGACAG	AACTTCAGGGTCAGCTTGCCG
GmActin	GGTGGTTCTATCTTGGCATC	CTTTCGCTTCAATAACCCTA

表2 大豆GmBEEs基因的外显子和内含子数量统计

Table 2 Statistics of the number of exons and introns of soybean GmBEEs genes

基因名 Gene name	基因ID Gene ID	定位标记 Locus tag	外显子个数 Number of exons	内含子个数 Number of introns
GmBEE3	100777160	Glyma08g042000	6	5
GmBEE1a	100810945	Glyma05g234500	6	5
GmBEE1b	100806942	Glyma09g183500	6	5
GmBEE1c	100807773	Glyma14g102200	6	5
GmBEE1d	100809974	Glyma17g223100	6	5
GmBEE1e	100803700	Glyma07g092700	6	5

图1 大豆GmBEEs基因家族的CDS序列比对

Fig. 1 CDS sequence alignment in soybean GmBEEs gene family

图2 大豆GmBEEs基因结构、基因保守结构域及基序分析A：GmBEEs基因结构； B：GmBEEs保守结构域； C：GmBEEs基序对比

Fig. 2 Analysis of gene structures, gene conserved domains and motifs of soybean GmBEEsA: Structure of GmBEEs gene. B: Conserved domains of GmBEEs. C: Gene motif alignment of GmBEEs

图3 大豆GmBEEs启动子顺式作用元件分析

Fig. 3 Analysis of cis-acting elements of soybean GmBEEs promoter

图4 大豆GmBEEs氨基酸的系统进化树

Fig. 4 Phylogenetic tree of soybeanGmBEEs amino acids

表3 大豆中5个GmBEE1和1个GmBEE3蛋白理化性质分析

Table 3 Analysis of physicochemical properties of five GmBEE1 and one GmBEE3 protein in soybean

基因名称 Gene name	基因ID Gene ID	氨基酸数 Number of amino acids	分子式 Formula	分子量 Molecular weight （Da）	等电点 pI
GmBEE1a	100810945	246	C₁₂₂₂H₁₉₂₆N₃₄₄O₃₈₁S₁₇	28 077.83	5.40
GmBEE1b	100806942	273	C₁₃₄₆H₂₁₂₆N₃₇₈O₄₁₆S₁₂	30 644.69	6.10
GmBEE1c	100807773	264	C₁₃₃₁H₂₀₇₅N₃₆₃O₄₀₈S₁₁	30 042.96	5.56
GmBEE1d	100809974	268	C₁₃₄₃H₂₀₈₆N₃₇₂O₄₁₉S₁₁	30 500.24	5.43
GmBEE1e	100803700	272	C₁₃₃₉H₂₁₁₅N₃₇₉O₄₁₃S₁₄	30 579.65	6.16
GmBEE3	100777160	252	C₁₂₂₆H₁₉₅₄N₃₄₆O₃₈₇S₁₆	28 246.05	5.53

图5 GmBEE1a和GmBEE3蛋白在烟草叶片亚细胞定位观察

Fig. 5 Subcellular localization of GmBEE1a and GmBEE3 in Nicotinan tabacum

图6 GmBEE1a和GmBEE3组织表达特异性误差线表示平均值±SD；***P<0.001； **P<0.01； *P<0.05；ns表示不存在显著性差异，下同

Fig. 6 Tissue-expression specifity of GmBEE1a and GmBEE3 in soybeanError bars indicate mean ± SD; ***P<0.001; **P<0.01; *P<0.05; ns indicates no significant difference, the same below

图7 大豆GmBEE1a和GmBEE3基因胁迫处理后表达情况

Fig. 7 Expressions of GmBEE1a and GmBEE3 genes in soybeans after stress treatment

图8 GmBEE1a和GmBEE3基因激素处理后表达情况

Fig. 8 Expressions of GmBEE1a and GmBEE3 genes after hormone treatment

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