大豆GS基因家族全基因组鉴定及胁迫响应分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0205

摘要/Abstract

摘要：

【目的】谷氨酰胺合成酶（glutamine synthetase, GS）是氮代谢“GS-GOGAT循环”中的关键酶，研究GS在大豆中的家族成员以及对外界胁迫的响应情况。【方法】利用生物信息学方法，在大豆中全面鉴定GS基因，明确大豆GmGSs基因的位置与结构、蛋白的理化性质以及组织表达模式等，并对非生物胁迫的应答响应进行研究。【结果】从大豆中共鉴定出8个GS基因，位于8条染色体上，对应编码的氨基酸序列长度为356-432 aa。在10个保守基序中，8个GmGS都包含9个保守基序，GmGS7和GmGS8比其他成员多一个motif10保守基序。启动子顺式作用元件分析表明，GmGSs的启动子中包含丰富的光响应元件、激素响应元件和逆境胁迫响应元件。转录组数据分析表明GmGSs基因在所有组织中均有表达，其中GmGS3和GmGS4在各组织中表达量较高。荧光定量qPCR结果表明：在不同浓度的氯化铵处理后，大豆GS家族中GmGS4、GmGS5和GmGS7对低浓度铵盐处理后期响应最显著。且高盐胁迫处理后，GmGS5在根、茎、叶组织中表达量下降；GmGS7在根、茎组织中表达量上升。【结论】GS在大豆中共有8个成员，其中GmGS7在氯化铵处理和盐胁迫中均参与响应。

关键词: 大豆, 谷氨酰胺合成酶, 生物信息学分析, 氯化铵, 盐胁迫

Abstract:

【Objective】Glutamine synthetase（GS）is a key enzyme in the GS-GOGAT cycle of nitrogen metabolism. This work is aimed to investigate the family members of GS in soybean and the response to external stress.【Method】Based on bioinformatics methods, we comprehensively identified GS genes in soybean（Glycine max）, clarified the location and structure of soybean GmGSs genes, the physicochemical properties of proteins, and the tissue expression patterns, and also studied the responses to abiotic stresses.【Result】The total of eight GS genes were identified from the soybean, located on eight chromosomes, corresponding to coding amino acid sequences with lengths ranging from 356 to 432 aa. Among the ten conserved motifs, all eight GmGS contained nine conserved motifs, and GmGS7 and GmGS8 had one more motif 10 conserved motif than other members. Analysis of promoter's cis-acting elements showed that the promoters of GmGSs contained abundant light-responsive elements, hormone-responsive elements, and adversity stress-responsive elements. Transcriptome data analysis showed that GmGSs genes were expressed in all tissues, with GmGS3 and GmGS4 having higher expression in all tissues. Fluorescence quantitative qPCR results revealed that GmGS4, GmGS5, and GmGS7 of soybean GS family responded most significantly to the late stage of low concentration ammonium treatment after different concentrations of ammonium chloride. And after high salt stress treatment, the expression of GmGS5 decreased in the root, stem and leaf tissues; while the expression of GmGS7 increased in root and stem tissues.【Conclusion】There are eight GS members in soybean, of which GmGS7 is involved in response to both ammonium chloride treatment and salt stress.

Key words: soybean, glutamine synthetase, bioinformatics analysis, ammonium chloride, salt stress

武帅, 辛燕妮, 买春海, 穆晓娅, 王敏, 岳爱琴, 赵晋忠, 吴慎杰, 杜维俊, 王利祥. 大豆GS基因家族全基因组鉴定及胁迫响应分析[J]. 生物技术通报, 2024, 40(8): 63-73.

WU Shuai, XIN Yan-ni, MAI Chun-hai, MU Xiao-ya, WANG Min, YUE Ai-qin, ZHAO Jin-zhong, WU Shen-jie, DU Wei-jun, WANG Li-xiang. Genome-wide Identification and Stress Response Analysis of Soybean GS Gene Family[J]. Biotechnology Bulletin, 2024, 40(8): 63-73.

图/表 10

表1 引物序列

Table 1 Primer sequence

基因ID Gene ID	正向引物 Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
GmCYP2	CGGGACCAGTGTGCTTCTTCA	CCCCTCCACTACAAAGGCTCG
GmGS1	TAACACCAAACAGAGTCATTCACC	TGAGGTTGATGAGATCCGAAA
GmGS2	GACTAATTTCCGGGGTTTCG	GGAGACCTTTTTCTTCCTCACAG
GmGS3	GCTTTTCTTAGTAGGATTTGGTCTC	TAACAATCGGAAAACGAGGGA
GmGS4	GTGGAAGCCATGAGCAAAACT	CGAGGGAAAGGAATAGAAAACA
GmGS5	TTGGAAACCATAAGCAGCCTC	GCCAAGCATTGAAGTGTGAGA
GmGS6	GCAACGTCAAAACAATCACATG	AACAACAGGCGAGGTAGTCACA
GmGS7	GCTGGTGTTGTGCTCTCTCT	TCTTCCCACACGGATTGAGC
GmGS8	ACCCAAAGGTCCAAGCAG	CCAGGATAGCCACCAACG

图1 大豆GS基因家族成员染色体分布

Fig. 1 Chromosome distribution of GS gene family in Gly-cine max

图2 大豆GS基因结构分析

Fig. 2 Analysis of GS gene structure in G. max

表2 大豆GS基因理化性质

Table 2 Physical and chemical properties analysis of GS genes in G. max

基因名称 Gene name	基因座位ID Gene locus ID	转录本ID Transcript ID	氨基酸数量Number of amino acids/aa	理论等电点Theoretical pI	分子量Molecular weight/kD	不稳定系Instability index	亲水平均数 Grand average of hydropathicity	亚细胞定位 Subcellular localization
GmGS1	Glyma.09g173200	KRH39032	356	5.22	39212.02	40.29	-0.467	Cytoplasmic
GmGS2	Glyma.07g104500	KRH48675	356	5.32	39148	40.49	-0.442	Cytoplasmic
GmGS3	Glyma.11g215500	KRH30930	356	5.48	38990.93	37.96	-0.374	Cytoplasmic
GmGS4	Glyma.18g041100	KRG97948	356	5.48	39140.98	39.47	-0.425	Cytoplasmic
GmGS5	Glyma.14g213300	KRH17324	356	6.12	39208.29	38.63	-0.444	Cytoplasmic
GmGS6	Glyma.02g244000	KRH72976	356	6.03	39326.47	39.75	-0.428	Cytoplasmic
GmGS7	Glyma.13g210800	KRH48676	432	6.42	47653.72	42.55	-0.466	Chloroplast
GmGS8	Glyma.15g102000	KRH30931	432	6.73	47691.83	43.16	-0.458	Chloroplast

图3 大豆GS基因保守结构域分析

Fig. 3 Conserved domain analysis of GS gene in G. max

图4 大豆GS基因系统进化树分析

Fig. 4 Phylogenetic tree analysis of GS gene in G. max

图5 大豆GS顺式作用元件分析

Fig. 5 Analysis of GS cis-acting elements in G. max

图6 大豆GS基因家族在不同组织（A）、不同氮处理（B）以及盐处理后（C）表达模式分析

Fig. 6 Expression patterns of G. max GS gene family in different tissues（A）, nitrogen treatment（B）, and salt treatment（C）

图7 大豆GS基因家族在铵盐处理后表达模式分析 1 d、3 d 分别表示处理后的1 d和3 d，CK、1 mmol、5 mmol分别表示氯化铵处理的浓度0 mmol/L、1 mmol/L、5 mmol/L。*、**、*** 分别表示与对照相比10%、5%、1%显著性水平，ns代表无显著性差异，下同

Fig. 7 Expression patterns of G. max GS family genes after ammonium salt treatment 1 d and 3 d indicate 1 and 3 days after treatment. CK, 1 mmol, and 5 mmol are the concentrations of ammonium chloride treatment, representing 0, 1 mmol/L, and 5 mmol/L, respectively. *, **, *** refers to 10%, 5% and 1% significance levels compared to control, and ns to no significant difference. The same below

图8 大豆GS基因家族在盐处理后表达模式分析 CK表示未处理对照，1、3、6、12、24、48表示150 mmol/L NaCI 处理后的时间，分别从左到右取样了GmGS5和GmGS7的根、茎、叶组织部位

Fig. 8 Expression patterns of G. max GS family genes after salt treatment CK indicates the untreated control, 1, 3, 6, 12, 24, 48 indicate the time after 150 mmol/L NaCI treatment, and the root, stem, and leaf tissue sites of GmGS5 and GmGS7 were sampled from left to right, respectively

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