大豆GmHMGS基因的鉴定及表达模式分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-1102

摘要/Abstract

摘要：

【目的】3-羟基-3-甲基戊二酰辅酶A合酶（HMGS）是甲羟戊酸途径中的一个关键酶，在植物生长发育和萜类化合物的合成中起着重要作用。对GmHMGS基因进行生物信息学和表达模式分析，为探究GmHMGS基因的功能奠定基础。【方法】运用生物信息学方法对该基因进行鉴定和分析，采用实时荧光定量PCR（RT-qPCR）对该基因表达模式进行分析。【结果】GmHMGS基因具有6个家族成员，命名为GmHMGS1-GmHMG6。理化性质和基因结构分析表明，GmHMGS3、GmHMG4、GmHMGS5、GmHMGS6为稳定的亲水性蛋白，GmHMGS都具有HMG-CoA合酶的保守结构域。启动子序列分析发现GmHMGS具有激素及逆境胁迫相关作用元件。组织表达模式分析发现，GmHMGS在根、茎、叶、花、籽粒、荚皮，根瘤中均有表达，GmHMGS1、GmHMG2、GmHMG3、GmHMG5、GmHMG6在叶片中的相对表达量较高，GmHMGS4在根中的相对表达量最高。非生物胁迫和不同外源激素诱导条件下表达分析表明，GmHMGS基因对PEG6000、NaCl、H₂O₂胁迫以及MeJA、ABA外源激素均有响应，且GmHMGS1和GmHMGS6在多种逆境胁迫下的相对表达量较高。【结论】GmHMGS基因家族可能参与大豆抗旱、耐盐和抗氧化胁迫的应答。

关键词: 大豆, GmHMGS, 生信分析, 表达分析, 胁迫处理

Abstract:

【Objective】3-hydroxy-3-methylglutaryl-CoA synthase（HMGS）is a key enzyme in the mevalonate pathway and plays an important role in the growth, development and synthesis of terpenoids. Bioinformatics study and expression pattern analysis of the GmHMGS gene can lay the foundation for exploring the function of GmHMGS gene. 【Method】The gene was identified and analysed using bioinformatics approaches, and then its expression pattern was examined using real-time fluorescence quantitative PCR（RT-qPCR）. 【Result】The GmHMGS gene had six family members in total, named GmHMGS1 to GmHMG6. The analysis of physicochemical properties and gene structure showed that GmHMGS3, GmHMG4, GmHMGS5, GmHMGS6 were stable hydrophilic proteins, and all GmHMGS possessed conserved functional domains of HMG-CoA synthase. Promoter sequence analysis revealed that GmHMGS has hormone and adversity stress related action elements. The analysis of tissue expression pattern revealed that GmHMGS was expressed in roots, stems, leaves, flowers, grains, pod skins, and root nodules, with leaves having the highest relative expression of GmHMGS1, GmHMG2, GmHMG3, GmHMG5, and GmHMG6, and roots having the highest relative expression of GmHMGS4. The GmHMGS genes were responsive to PEG6000, NaCl, and H₂O₂ stresses, as well as MeJA and ABA exogenous hormones, and the relative expression of GmHMGS1 and GmHMGS6 was higher under a wide range of adversity stresses, according to expression analysis under abiotic stress and different exogenous hormone-induced conditions. 【Conclusion】GmHMGS gene family may be involved in response to drought, oxidative and salt stresses in soybean.

Key words: soybean, GmHMGS, biocredit analysis, expression analysis, stress treatment

娄银, 高浩竣, 王茜, 牛景萍, 王敏, 杜维俊, 岳爱琴. 大豆GmHMGS基因的鉴定及表达模式分析[J]. 生物技术通报, 2024, 40(4): 110-121.

LOU Yin, GAO Hao-jun, WANG Xi, NIU Jing-ping, WANG Min, DU Wei-jun, YUE Ai-qin. Identification and Expression Pattern Analysis of GmHMGS Gene in Soybean[J]. Biotechnology Bulletin, 2024, 40(4): 110-121.

图/表 11

表1 实时荧光定量引物序列

Table 1 Primers sequences for real-time quantitative PCR

基因名称Gene name	登录号Gene ID	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
GmActing	XM_003552652	GGTGGTTCTATCTTGGCATC	CTTTCGCTTCAATAACCCTA
GmHMGS1	Glyma.01G215500.1	GGAGGAACTGCTGCTCTGTT	GGGCAGGTCCTTCAGCATAG
GmHMGS2	Glyma.05G050300.1	TCCAGTGGTTGATGGGAAGC	CCTCATCCACAAAACTGGCAT
GmHMGS3	Glyma.09G148500.1	GAGTAGCTCATGGGATGGACG	TAGGAGCATCTGGCCCTACA
GmHMGS4	Glyma.11G027000.1	AACATGTACACGGCGTCTCT	ATGGTGGCAGTTAAGCCACT
GmHMGS5	Glyma.16G200200.1	ATGCTATGGAGGAACGGCTG	ATCTGGCCCTACAAGCATGG
GmHMGS6	Glyma.17G132300.1	CCAGCAAGTTGCAAAGCCTC	GATGCGGTGTACATGTTGCC

表2 GmHMGS基因序列信息和理化性质分析

Table 2 Analysis of GmHMGS gene sequence information and physicochemical properties

基因名称 Gene name	基因ID Gene ID	基因全长 Gene length/bp	CDS序列 CDS sequence/bp	氨基酸数 Amino acid amounts	分子式 Molecular formula	分子量 Mw/kD	等电点 PI	不稳定系数 Instability index	亲水性平均值 GRAVY
GmHMGS1	Glyma.01G215500.1	5 336	1 416	471	C₂₃₄₀H₃₆₀₄N₆₀₆O₇₀₇S₂₃	72.80	6.20	40.17	-0.231
GmHMGS2	Glyma.05G050300.1	4 665	882	293	C₁₄₃₃H₂₂₂₃N₃₇₇O₄₃₃S₁₂	44.78	6.08	41.69	-0.178
GmHMGS3	Glyma.09G148500.1	6 211	1 374	457	C₂₂₆₆H₃₄₈₈N₅₈₄O₆₈₂S₂₅	70.45	5.90	39.37	-0.163
GmHMGS4	Glyma.11G027000.1	6 456	1 428	475	C₂₃₄₄H₃₆₂₅N₆₀₅O₇₁₈S₂₂	73.14	5.77	39.33	-0.224
GmHMGS5	Glyma.16G200200.1	6 465	1 365	454	C₂₂₃₈H₃₄₄₀N₅₇₈O₆₈₀S₂₅	69.61	5.78	36.97	-0.174
GmHMGS6	Glyma.17G132300.1	4 760	1 383	460	C₂₂₈₇H₃₅₃₄N₅₈₈O₆₉₄S₂₂	71.25	5.98	39.33	-0.246

图1 GmHMGS基因结构分析

Fig. 1 Analysis of GmHMGS gene structure

表3 GmHMGS的二级结构预测及亚细胞定位预测

Table 3 Prediction of secondary structure and subcellular localization of GmHMGS

蛋白名称Protein name	α螺旋Alpha helix/%	延伸链Extended strand/%	无规则卷曲Random coil/%	染色体Chr.	亚细胞定位Subcellular localization
GmHMGS1	25.05	22.29	52.65	01	细胞核Nucleus
GmHMGS2	25.26	22.53	52.22	05	细胞核Nucleus
GmHMGS3	22.32	19.91	57.77	09	叶绿体Chloroplast
GmHMGS4	23.37	21.89	54.74	11	细胞核Nucleus
GmHMGS5	21.81	21.59	56.61	16	叶绿体Chloroplast
GmHMGS6	31.09	20.87	48.04	17	细胞核Nucleus

图2 GmHMGS蛋白的三级结构

Fig. 2 Tertiary structure of GmHMGS protein

图3 GmHMGS保守基序分析 A：GmHMGS保守元件分析，Motif 1-6为不同颜色代表的基序组成；B：GmHMGS蛋白的保守基序序列

Fig. 3 Motif composition of the CmHMGS gene A: GmHMGS conserved element analysis, Motif 1-6 in different colored blocks indicate the motif composition, B: Sequence of conserved structural elements of GmHMGS proteins

图4 GmHMGS 基因的启动子顺式作用元件分析 ABRE：脱落酸响应元件；ARFs：生长素响应元件；GARE-motif：赤霉素响应元件；G-box：光响应元件；TCA-element：水杨酸响应元件； MBS：干旱胁迫响应元件；ARE：厌氧诱导元件；LTR：低温响应元件；HSE：胁迫响应元件

Fig. 4 Analysis of promoter cis-acting elements of the GmHMGS gene ABRE: Abscisic acid response element; ARFs: growth factor response elements; GARE-motif: gibberellin response element; G-box: light-responsive element; TCA-element: salicylic acid response element; MBS: Drought stress response element; ARE: anaerobic recovery element; LTR: low temperature response element; HSE: stress response element

图5 GmHMGS蛋白系统进化分析黄芪（Astragalus membranaceus, Am）；籼稻（Oryza sativa, Os）；小米（Setaria italica, Si）；玉米（Zea mays, Zm）；二穗短柄草（Brachypodium distachyon, Bd）；拟南芥（Arabidopsis thaliana, At）；芥菜（Brassica juncea, Bj）；雷蒙德氏棉（Gossypium raimondii, Gr）；烟草（Nicotiana sylvestris, Ns）；番茄（Solanum lycopersicum, Sl）；朗氏烟草（Nicotiana sanderae, Ns）；芝麻（Sesamum indicum, Si）；丹参（Salvia miltiorrhiza, Sm）；薰衣草（Lavandula angustifolia, La）；桔梗（Platycodon grandifloras, Pg）；三七（Panax notoginseng, Pn）；黄花蒿（Artemisia annua, Aa）；橡胶树（Hevea brasiliensis, Hb）；蓖麻（Ricinus communis, Rc）；苹果（Malus domestica, Md）；桃（Prunus persica, Pp）；红豆杉（Taxus media, Tm）；轮藻（Chara vulgaris, Cv）

Fig. 5 Analysis of the phylogenetic evolution of GmHMGS proteins

图6 GmHMGS基因家族在不同组织中的表达分析不同字母表示 P <0.05 水平上有显著差异

Fig. 6 Expression analysis of the GmHMGS gene family in different tissues Different letters indicate significant differences at the P <0.05 level

图7 非生物胁迫下大豆GmHMGS基因的表达模式分析 A：PEG6000 处理；B：NaCl 胁迫；C：H2O2 胁迫；对照组为处理后0 h GmHMGS基因表达水平设定为1，相应地计算处理GmHMGS的基因表达水平；数据为 3 个重复的平均值 ± SE；不同字母表示在 P <0.05 水平上有显著差异

Fig. 7 Expression patterns of GmHMGS gene in response to abiotic stress in soybean A: PEG6000 treatment; B: NaCl stress; C: H2O2 stress. The gene expression level of GmHMGS of the control at 0 h post-treatment was set to 1, and those of GmHMGS of the treatments were accordingly accounted and presented as the relative fold changes. The data indicate the mean ± SE of three replicates. Different letters indicate significant differences at the P <0.05 level

图8 激素处理后大豆GmHMGS基因的表达模式分析 A：MeJA处理；B：ABA处理；对照组为处理后0 h GmHMGS基因表达水平设定为1，相应地计算处理GmHMGS的基因表达水平；数据为3 个重复的平均值 x ± SE；不同字母表示在 P <0.05 水平上有显著差异

Fig. 8 Expression patterns of GmHMGS gene in response to hormone treatment in soybean A: MeJA treatment; B: ABA treatment. the gene expression level of GmHMGS of the control at 0 h post-treatment was set to 1, and those of GmHMGS of the treatments were accordingly accounted and presented as the relative fold changes. The data indicate the mean x±SE of three replicates. Different letters indicate significant differences at the P <0.05 level

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