Genome-wide identification and tissue expression analysis of IGT gene family in soybean

CHEN Mengyao; HU Yiran; ZHENG Zhifu; PAN Tian

doi:10.11833/j.issn.2095-0756.20240354

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CHEN Mengyao, HU Yiran, ZHENG Zhifu, PAN Tian. Genome-wide identification and tissue expression analysis of IGT gene family in soybean[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20240354

Citation:

CHEN Mengyao, HU Yiran, ZHENG Zhifu, PAN Tian. Genome-wide identification and tissue expression analysis of IGT gene family in soybean[J]. Journal of Zhejiang A&F University. doi: 10.11833/j.issn.2095-0756.20240354

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Genome-wide identification and tissue expression analysis of IGT gene family in soybean

doi: 10.11833/j.issn.2095-0756.20240354

College of Advanced Agricultural Sciences, Zhejiang A&F University, Hangzhou 311300, Zhejiang, China

Received Date: 2024-05-15
Accepted Date: 2024-08-09
Rev Recd Date: 2024-08-06

Abstract

Objective This study aims to employ bioinformatics methods to perform a comprehensive genome-wide identification of the soybean IGT gene family and explore the potential functions of IGT gene family in soybean. Method Soybean cultivar Glycine max‘Tianlong 1’ was selected and GmIGTs were identified by BLASTP search of IGT protein domain. The evolutionary relationships, gene structure, conserved motifs, cis-acting elements, and collinearity relationships were analyzed by bioinformatics methods. The expression patterns of GmIGTs in different tissue parts were analyzed. Result A total of 17 GmIGT genes were identified and classified into 4 branches based on their phylogenetic relationships: TAC, IGT-like, DRO, and LAZY. Protein conserved motif analysis revealed that all IGT proteins contained Motif2. Chromosomal localization and collinearity analysis showed that GmIGT genes were unevenly distributed on 11 chromosomes, and segmental duplication might have played a significant role in the expansion of GmIGT gene. Cis-acting element analysis indicated that GmIGT expression might be associated with light response, physiological response, plant hormone response and stress. RT-qPCR analysis demonstrated that GmIGT gene had obvious tissue-specific expression characteristics. Among them, GmIGT5 exhibited relatively high expression levels in all tissues, while GmIGT4 and GmIGT10 were highly expressed in stems and petioles. Conclusion GmIGT gene may play a potential role in shaping soybean plant architecture, and GmIGT4 and GmIGT10 may be the core genes involved in this process. [Ch, 9 fig. 2 tab. 23 ref.]
- Glycine max ‘Tianlong 1’,
- plant architecture,
- IGT gene family,
- expression pattern analysis

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优化植物株型已被证明是提高作物种植密度、抗逆性和整体生产力的最佳方法之一^[1]。随着基因组学的发展，科学家们发现了被命名为IGT的保守基因家族，与植物分枝角度调控密切相关^[2]。这个基因家族拥有1个保守的结构域[GϕL(A/T)IGT]，包含着LAZY和TILLER ANGLE CONTROL (TAC)基因。这些基因在植物侧枝的遗传控制中扮演着重要角色^[3]。

近几十年来的研究已经确认，LAZY基因在植物根部的向地生长和茎部的背地生长中起到了重要的调节作用。“LAZY”这个术语来源于被认为是“懒惰”的经典水稻Oryza sativa和玉米Zea mays植株，它们的特点在水稻中表现为分蘖扩散，而在玉米中表现为平卧^[4−5]。LI等^[6]通过研究水稻LAZY变异体，成功克隆了1个名为LAZY1的基因，该基因调控着分蘖角度。拟南芥Arabidopsis thaliana中也发现存在LAZY1突变体，被命名为AtLAZY1。拟南芥分枝朝外生长，并且比野生型植株的分枝角度更宽^[3]。LAZY基因还被命名为其他名称，如DEEPER ROOTING(DRO)。水稻基因组携带有4个LAZY基因，分别是LAZY1、DRO1、DRO1-like 1(qSOR1, DRL1)和DRO1-like 2(DRL2)^[7]。DRO1和DRL1的自然突变以及DRL2的CRISPR/Cas9技术产生的敲除突变都导致根部重力性反应受损，根系结构也发生改变，甚至出现裸露在地面的根^[8]。GE等^[9]在拟南芥中发现了3个类似DRO1的基因：AtDRO1(AtNGR2，At1g72490)、AtDRO2(AtNGR3，At1g19115)和AtDRO3(AtNGR1，At1g17400)。这3个基因都具有最保守的LAZY1结构域Ⅴ，即EAR基序，因而它们又分别被命名为AtLAZY4、AtLAZY3、AtLAZY2；相应地，At3g24750和At3g27025分别被命名为AtLAZY5和AtLAZY6。它们分别在根尖、根和叶柄中表达^[10]。YU等^[11]以水稻‘IR24’和‘Asominori’杂交，获得‘IL55’紧凑型杂交系，并成功定位克隆到水稻分蘖角度控制基因TAC1(TILLER ANGLE CONTROLLING)。TAC1基因增加了分枝角度，与LAZY1基因突变植物表型相反。研究表明：AtTAC1的表达具有光依赖性^[12]，并且在拟南芥中，几个LAZY基因的缺失导致其无法调整枝条角度以响应光照^[13]。这些研究表明：IGT基因家族在响应光和重力刺激的植物株型调节中发挥作用。

大豆Glycine max是重要的粮食和油料作物，营养成分丰富。当前，大豆为全球供应了71%的植物蛋白和29%的植物油脂^[14−16]。然而2022年中国进口大豆数量达到了9 108万t，对外依存度高达80%以上，大豆产需缺口高达9 000万t。因此，优化大豆株型对提高中国大豆种植密度以及提升中国大豆产量有着重要意义。IGT基因家族作为株型调控的潜在基因，在大豆中的系统鉴定和功能尚不明确。本研究利用生物信息学方法对大豆IGT基因家族(GmIGTs)进行全基因组水平鉴定，并对IGT基因家族成员进化关系、基因结构、保守基序、顺式作用元件、共线性关系及组织表达量等进行分析，以期为深入探究IGT基因家族在大豆生长发育过程中的作用机制奠定理论基础。

1. 材料与方法

1.1. 大豆IGT基因家族成员鉴定和进化分析

在Phytozome (https://phytozome-next.jgi.doe.gov/)和美国国家生物技术信息中心(NCBI)数据库(https://www.ncbi.nlm.nih.gov)对大豆‘天隆1号’‘Tianlong 1’ IGT基因家族氨基酸序列与拟南芥进行BLAST比对，确认大豆IGT基因家族成员，收集大豆基因组数据(www.soybase.org)中的大豆IGT基因家族成员遗传信息，并利用蛋白质数据库分析大豆IGT基因家族成员基本信息。

在Phytozome (https://phytozome-next.jgi.doe.gov/)下载拟南芥以及大豆IGT基因家族氨基酸序列，对氨基酸序列进行多重比对。采用最大似然法构建进化树(MEGA 5.0软件)。

1.2. GmIGT基因结构、蛋白质保守基序分析

下载大豆基因组Phytozome (https://phytozome-next.jgi.doe.gov/)，使用TBtools 对IGT基因家族外显子-内含子结构分析，并绘制可视化图。同时，对大豆IGT基因家族进行蛋白质保守基序研究(MEME)，利用TBtools进行可视化绘图。

1.3. GmIGT启动子区域顺式作用元件分析

提取大豆IGT基因家族上游2 000 bp序列，并利用PlantCARE数据库对启动子区域的顺式作用元件进行分析。随后，使用在线工具GSDS (http://gsds.gao-lab.org/)对结果进行可视化处理。

1.4. GmIGT共线性分析

通过Phytozome (https://phytozome-next.jgi.doe.gov/)下载大豆的全基因组文件，利用 TBtools软件进行共线性分析，并进一步可视化。

1.5. GmIGT组织表达模式预测和分析

通过从Phytozome (https://phytozome-next.jgi.doe.gov/)数据库下载的大豆在不同组织的基因表达数据，选择分生组织、根、茎、叶、花、荚、种子和根瘤等8个组织的基因表达数据进行热图分析，分析IGT基因家族在大豆中的表达情况。采用TBtools 热图软件进行数据可视化处理。

采用TransZol Up Plus RNA Kit (ER501，Transgene)提取大豆的根、茎、叶、叶柄、花、果荚、种子、根瘤各部位RNA，采用Hifair® Ⅲ 1 st Strand cDNA Synthesis SuperMix for qPCR (11141ES60, 翌圣)进行反转录合成 cDNA，所得的cDNA用于检测GmIGT基因的表达水平。在Phytozome (https://phytozome-next.jgi.doe.gov/)上检索获得IGT基因的序列，以大豆cons4为内参基因，使用Snap Gene软件设计跨内含子的上下游引物(表1)，由擎科生物技术有限公司(杭州)合成。将大豆不同组织(根、茎、叶、叶柄、花、果荚、种子、根瘤)的cDNA质量浓度定量至50 mg·L⁻¹，按照Hieff UNICON® qPCR SYBR Green Master Mix (抗体法，No Rox，11198ES08, 翌圣)操作说明进行实时荧光定量PCR(RT-qPCR)。每个样品含3个生物学重复。利用$2^{-\Delta \Delta C_{\mathrm{T}}} $法计算表达水平^[17]。

引物名称	引物序列(5′→3′)	引物名称	引物序列(5′→3′)
IGT1-qRT-F	TTGGTCATTGGGAATTCTTGCAA	IGT9-qRT-R	CAATCGCTGAATTCTTCCCT
IGT1-qRT-R	CTTCTACACCAGCAAAAGAGT	IGT10-qRT-F	GAAAGATGGGTTTGCTACAAAC
IGT2-qRT-F	ATGCAATTCCTCAGCTGGA	IGT10-qRT-R	CCGAGAGTGCCAATGGTT
IGT2-qRT-R	TGCTAGTAAACCATCAGGC	IGT11-qRT-F	TTCACACTTGGAAACCCTTGTA
IGT3-qRT-F	AAGTTCCTCAGCTGGATG	IGT11-qRT-R	CAACCCAGAGTAAGATGTTTGA
IGT3-qRT-R	GGCCAATCGCTGAATTC	IGT12-qRT-F	ATGCAGATTCTTCAATGGATCTCC
IGT4-qRT-F	CTGAAAGATGGGTTTGCTTCA	IGT12-qRT-R	CCTCCCTTCGATTGTCTTT
IGT4-qRT-R	GATCATAGCCAAGAGTGCCAA	IGT13-qRT-F	CAGTGAACAAAATGAAGGGAAAG
IGT5-qRT-F	TCAGTGGGAAAGAGGTAAATC	IGT13-qRT-R	GTTGAGGGTGCTGTAAAAGAA
IGT5-qRT-R	CTGAACATTAAAACCTTTCTTCCA	IGT14-qRT-F	CTTGGTCATTGGGAATTCTTGC
IGT6-qRT-F	GAATGGAAAAGATTTTGAGGGCAATAC	IGT14-qRT-R	CCAGCAAAAGAGTTTCTAAGGT
IGT6-qRT-R	AAGCTCCTCTTCATCATCATC	IGT15-qRT-F	GATTTCACACTTGGAAATCCCTG
IGT7-qRT-F	TTGATCATCGGAGGGAATTCTT	IGT15-qRT-R	CCCAGAGTAAGATGTTTGATTTTCC
IGT7-qRT-R	CTGAATCTTCCTCATACTCATCATG	IGT16-qRT-F	AAATGATCATGAAATAGGCCGG
IGT8-qRT-F	CAACTACCCATTACATGAGGC	IGT16-qRT-R	TCCTCCCTTCGATTGTCTTT
IGT8-qRT-R	GCGTGGTTACTCCTTCCA	IGT17-qRT-F	TTGATCATCGGAGGAGGGA
IGT9-qRT-F	CAATGAAGTTCCTCAGCTGGAT	IGT17-qRT-R	CTTCCTCATACTCATCATGATCC

Table 1. Primer sequences for RT-qPCR

3. 讨论

IGT基因家族在重力感应中起着关键作用，因此推测该基因家族参与植物株型的调控。IGT基因家族广泛存在于植物基因组中，且在不同物种中的数量差距较大。目前，已在水稻、玉米、油菜Brassica campestris和苹果Malus domestica中分别鉴定出4、2、27和4个IGT基因家族成员^{[1，18−20]}。本研究在深入分析大豆基因组基础上，鉴定得到17个大豆IGT基因家族成员，它们不均匀分布在11条染色体上。其基因总数与其他物种中IGT基因家族成员数目有所差异，这可能与不同物种间基因组大小、基因扩张程度不同有关。基因组内共线性分结果表明：GmIGT复制类型主要为片段性复制，有14条片段复制事件，这说明片段性复制在GmIGT基因扩张中起了至关重要的作用。这些家族成员的扩增可能会导致不同成员产生功能冗余性或专化性，从而为生物过程的精细调控提供基础。

在拟南芥中，IGT基因家族存在5个较短的保守结构域和1个独特的内含子排列^[1]，GmIGT基因中也出现了类似的内含子排列。外显子和内含子排列可在一定程度上解释该基因家族的进化史。在很多情况下，不同物种中具有相似基因结构和保守基序的同源蛋白可能具有相同或相似的功能。由此推测，犹如拟南芥AtIGT基因，大豆GmIGT可能参与大豆的重力传感和植物株型的形成。

前人研究表明：LAZY基因往往在拟南芥、水稻、蒺藜Tribulus terrestris和日本百里香Thymus vulgaris等高等植物中形成1个小基因家族^{[3, 21]}。在拟南芥中，该基因家族有6个成员，不同成员在不同器官中的功能可能存在差异，已知AtLAZY1调节芽和花序茎的向地性^{[3, 10]}，但AtLAZY5、AtLAZY6在向地性方面的功能尚未见报道。在许多植物中，TAC在调节分枝角度方面起着重要作用，OsTAC1参与水稻分蘖角度的调节，并影响内源生长素的含量^[22]。在玉米中，ZmTAC1的表达水平与叶片角度呈正相关，该基因在叶片发育中起着重要的调控作用^[23]。系统发育分析显示：IGT基因家族在大豆中可以分为4个进化枝，TAC、IGT-like、DRO以及LAZY进化枝。同时，LAZY、IGT-like、DRO进化枝的成员并不都具有完全相同的Motif，这可能意味着其进化枝上的基因在功能上存在差异，这种基序的差异可能反映了这些进化枝在生物学功能或调控机制上的不同。因此，根据聚类结果，大豆的IGT基因家族可能在不同的器官中起着不同的作用，但其作用有待进一步研究。

根据启动子预测结果，IGT基因家族存在光响应、生长发育、激素响应及胁迫响应元件，表明其可能受到光照、植物激素的调控诱导或参与其信号反应途径，同时还可能参与大豆生长发育的某些过程以及参与胁迫过程中的抗逆调控。

通过数据库预测，GmIGT基因成员在不同的组织和器官中显示出特异性的表达模式， RT-qPCR分析结果与这些基因表达模式的预测结果基本一致。组织特异性表达分析发现：整体相对表达量最高的为GmIGT4，推测该基因可能在很多组织、器官中发挥作用；GmIGT4、GmIGT10和GmIGT17在茎和叶柄中的相对表达量显著高于其他组织，推测其可能在大豆株型调控方面发挥重要作用。不同IGT基因家族成员在大豆不同组织中表达模式的差异暗示了其可能在根的向地性调节、茎的生长发育等方面存在功能上的分化，这为进一步解析GmIGT基因在大豆生长发育和株型形成中的作用奠定基础。

4. 结论

本研究对大豆IGT基因家族从全基因组层面进行了全面的生物信息学分析，共鉴定出17个GmIGT，分为TAC、IGT-like、DRO以及LAZY等4个进化枝。表达模式分析显示：GmIGT基因成员在大豆不同组织中表达模式具有差异性，GmIGT4、GmIGT10和GmIGT17可能在塑造大豆株型中发挥主要作用。该结果为进一步了解GmIGT基因在塑造植物株型中的生物学功能提供了理论基础。

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基因名称	序列号	染色体	位置	碱基对/bp	预测蛋白质长度/aa	理论等电点	分子量/Da	亚细胞定位
GmIGT1	Glyma.02G097100	Chr2	8677685..8680757	1 101	367	6.91	40 326.05	细胞核
GmIGT2	Glyma.03G264200	Chr3	46860193..46864806	822	274	5.52	31 485.80	细胞核
GmIGT3	Glyma.07G040800	Chr7	3391340..3394088	846	282	7.58	32 278.71	叶绿体
GmIGT4	Glyma.07G052500	Chr7	4555925..4560419	858	286	5.45	32 189.21	细胞核
GmIGT5	Glyma.08G356200	Chr8	46252966..46256085	852	284	10.03	32 316.57	细胞核
GmIGT6	Glyma.09G071100	Chr9	7218596..7221206	789	263	5.11	29 050.36	细胞核
GmIGT7	Glyma.10G298100	Chr10	51546352..51548699	1068	356	5.96	39 907.67	细胞质
GmIGT8	Glyma.15G179200	Chr15	17222927..17225610	789	263	5.12	29 253.52	细胞核
GmIGT9	Glyma.16G009400	Chr16	842813..845650	855	285	8.03	32 507.05	细胞核
GmIGT10	Glyma.16G021400	Chr16	2005820..2009925	870	290	5.45	32 743.91	细胞质
GmIGT11	Glyma.16G054300	Chr16	5286678..5290902	1 149	383	6.65	43 362.59	叶绿体
GmIGT12	Glyma.16G094000	Chr16	15479890..15482382	786	262	9.67	30 379.48	细胞核
GmIGT13	Glyma.18G173300	Chr18	41543428..41546170	861	287	9.92	32 629.87	细胞核
GmIGT14	Glyma.18G284400	Chr18	56775415..56778618	1 098	366	6.91	40 390.19	细胞核
GmIGT15	Glyma.19G094600	Chr19	33952844..33956542	1 149	383	7.18	43 382.51	细胞核
GmIGT16	Glyma.19G263200	Chr19	51104292..51108788	807	269	5.68	30 957.17	细胞核
GmIGT17	Glyma.20G249200	Chr20	47749661..47752260	1 080	360	5.87	40 051.88	细胞核