Cloning and Functional Identification of Drought Responsive G-box Binding Factor <i>GmDIBL1</i> in Soybean

QIAO Zhiqing; QIU Yushan; GAO Yinghui; LI Yangcun; LIU Fei; ZHANG Hongju; TIAN Huidan; WANG Qiaoling; LIU Ning; TAN Pingting; LIU Chen; LUO Ming

doi:10.13718/j.cnki.xdzk.2024.09.003

2024 Volume 46 Issue 9

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QIAO Zhiqing, QIU Yushan, GAO Yinghui, et al. Cloning and Functional Identification of Drought Responsive G-box Binding Factor GmDIBL1 in Soybean[J]. Journal of Southwest University Natural Science Edition, 2024, 46(9): 35-44. doi: 10.13718/j.cnki.xdzk.2024.09.003

Citation:

QIAO Zhiqing, QIU Yushan, GAO Yinghui, et al. Cloning and Functional Identification of Drought Responsive G-box Binding Factor GmDIBL1 in Soybean[J]. Journal of Southwest University Natural Science Edition, 2024, 46(9): 35-44. doi: 10.13718/j.cnki.xdzk.2024.09.003

Cloning and Functional Identification of Drought Responsive G-box Binding Factor GmDIBL1 in Soybean

1.
College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
2.
Engineering Research Center of South Upland Agriculture, Ministry of Education, Chongqing 400715, China
3.
College of Life Sciences, Southwest University, Chongqing 400715, China

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Corresponding author: LUO Ming
Received Date: 14/06/2024
Available Online: 20/09/2024
MSC: S565.1

Abstract

Drought is one of the typical abiotic stresses that seriously affect the yield and quality of crops. Identifying the functions of key genes for the drought resistance/tolerance of crop and analyzing their regulatory mechanisms are of great significance for developing new drought resistant germplasm of crops and ensuring food security. Soybean is a crop that is more sensitive to drought stress. Exploring and searching for soybean drought resistance/tolerance genes, identifying their functions, and revealing their regulatory mechanisms are hot topics in the field of soybean stress resistance research. This study analyzed transcriptome data of drought treated soybean roots and obtained a drought induced G-box binding protein gene GmDIBL1 (Drought Induced B12D Like 1). The cloned cDNA contained an open reading frame of 261 bp length, which encoded 86 amino acid residues. The molecular weight and isoelectric point of GmDIBL1 was 10.1 kD and 9.647, respectively. The GmDIBL1 protein sequence exhibits a high conservation with G-box binding proteins from other species, including the B12D domain (6-73 amino acid residues) and transmembrane helix. The N-terminus has a signal peptide, while the C-terminus forms a non-cytoplasmic region (34-86 amino acid residues). GmDIBL1 is located in the nucleus. The expression level of GmDIBL1 gene in root nodules is the highest, followed by roots. After drought treatment for 12 h, GmDIBL1 expression level significantly increased. The expression level of GmDIBL1 was much higher in the root of 'Jindou 21' (a drought resistant variety) than in that of 'Suinong 26' (a drought sensitive variety). Furthermore, the response of this gene to drought was identified by overexpressing and knocking out the GmDIBL1 gene in soybean. Overexpressing GmDIBL1 gene significantly enhanced the drought resistance of the hairy roots complex. These results indicated that the GmDIBL1 gene is a drought stress responsive gene in soybeans and plays an important role in resistance/tolerance to drought stress.
- drought response,
- soybean,
- G-box binding protein,
- GmDIBL1 gene,
- cloning,
- hairy roots,
- gene editing

References

[1]	任洪雷, 朱筱, 张丰屹, 等. 干旱胁迫的影响及抗旱性研究进展[J/OL]. 分子植物育种, 2024: 1-10. (2024-01-22)[2024-03-01]. https://kns.cnki.net/kcms/detail/46.1068.S.20240119.1548.002.html. Google Scholar
[2]	SHAHEEN T, RAHMAN M U, SHAHIDRIAZ M, et al. Soybean Production and Drought Stress[M] //Abiotic and Biotic Stresses in Soybean Production. Amsterdam: Elsevier, 2016: 177-196. Google Scholar
[3]	SPECHT J E, HUME D J, KUMUDINI S V. Soybean Yield Potential-A Genetic and Physiological Perspective[J]. Crop Science, 1999, 39(6): 1560-1570. doi: 10.2135/cropsci1999.3961560x CrossRef Google Scholar
[4]	朱筱, 张丰屹, 马晓宇, 等. 大豆抗旱性的生理和分子机制及其在遗传改良中的应用[J/OL]. 分子植物育种, 2023: 1-11. (2023-04-23)[2024-03-01]. https://kns.cnki.net/kcms/detail/46.1068.S.20230421.1701.016.html. Google Scholar
[5]	GIULIANO G, PICHERSKY E, MALIK V S, et al. An Evolutionarily Conserved Protein Binding Sequence Upstream of a Plant Light-Regulated Gene[J]. Proceedings of the National Academy of Sciences of the United States of America, 1988, 85(19): 7089-7093. Google Scholar
[6]	ALMUTAIRI Z M. Expression Profiling and Characterization of a G-box Binding Protein, B12Dg, from Pearl Millet[J]. Journal of King Saud University-Science, 2023, 35(1): 102448. doi: 10.1016/j.jksus.2022.102448 CrossRef Google Scholar
[7]	SCHINDLER U, BECKMANN H, CASHMORE A R. TGA1 and G-box Binding Factors: Two Distinct Classes of Arabidopsis Leucine Zipper Proteins Compete for the G-box-like Element TGACGTGG[J]. The Plant Cell, 1992, 4(10): 1309-1319. Google Scholar
[8]	STEINUM T M, BERNER H S, STACY R A P, et al. Differential Regulation of the Barley (HordeumVulgare) Transcripts B22E and B12D in Mature Aleurone Layers[J]. Physiologia Plantarum, 1998, 102(3): 337-345. doi: 10.1034/j.1399-3054.1998.1020301.x CrossRef Google Scholar
[9]	AALEN R B, SALEHIAN Z, STEINUM T M. Stability of Barley Aleurone Transcripts: Dependence on Protein Synthesis, Influence of the Starchy Endosperm and Destabilization by GA3[J]. Physiologia Plantarum, 2001, 112(3): 403-413. doi: 10.1034/j.1399-3054.2001.1120314.x CrossRef Google Scholar
[10]	HE D L, ZHANG H, YANG P F. The Mitochondrion-Located Protein OsB12D1 Enhances Flooding Tolerance during Seed Germination and Early Seedling Growth in Rice[J]. International Journal of Molecular Sciences, 2014, 15(8): 13461-13481. doi: 10.3390/ijms150813461 CrossRef Google Scholar
[11]	ZHOU Y Q, ZHANG Y H, WEI J, et al. Cloning and Analysis of Expression Patterns and Transcriptional Regulation of RghBNG in Response to Plant Growth Regulators and Abiotic Stresses in Rehmannia glutinosa[J]. Springer Plus, 2015, 4: 60. doi: 10.1186/s40064-015-0830-0 CrossRef Google Scholar
[12]	AALEN R B. The Transcripts Encoding Two Oleosin Isoforms are both Present in the Aleurone and in the Embryo of Barley (Hordeum vulgare L.) Seeds[J]. Plant Molecular Biology, 1995, 28(3): 583-588. doi: 10.1007/BF00020404 CrossRef Google Scholar
[13]	HUANG Y J, TO K Y, YAP M N, et al. Cloning and Characterization of Leaf Senescence Up-Regulated Genes in Sweet Potato[J]. Physiologia Plantarum, 2001, 113(3): 384-391. doi: 10.1034/j.1399-3054.2001.1130312.x CrossRef Google Scholar
[14]	ZHU T, BUDWORTH P, HAN B, et al. Toward Elucidating the Global Gene Expression Patterns of Developing Arabidopsis: Parallel Analysis of 8 300 Genes by a High-Density Oligonucleotide Probe Array[J]. Plant Physiology and Biochemistry, 2001, 39(3-4): 221-242. doi: 10.1016/S0981-9428(00)01234-1 CrossRef Google Scholar
[15]	ERGEN N Z, THIMMAPURAM J, BOHNERT H J, et al. Transcriptome Pathways Unique to Dehydration Tolerant Relatives of Modern Wheat[J]. Functional & Integrative Genomics, 2009, 9(3): 377-396. Google Scholar
[16]	WANG Y G, STEVANATO P, LV C H, et al. Comparative Physiological and Proteomic Analysis of Two Sugar Beet Genotypes with Contrasting Salt Tolerance[J]. Journal of Agricultural and Food Chemistry, 2019, 67(21): 6056-6073. doi: 10.1021/acs.jafc.9b00244 CrossRef Google Scholar
[17]	MOHANTY B. Promoter Architecture and Transcriptional Regulation of Genes Upregulated in Germination and Coleoptile Elongation of Diverse Rice Genotypes Tolerant to Submergence[J]. Frontiers in Genetics, 2021, 12: 639654. doi: 10.3389/fgene.2021.639654 CrossRef Google Scholar
[18]	ALMUTAIRI Z M. In Silico Identification and Characterization of B12D Family Proteins in Viridiplantae[J]. Evolutionary Bioinformatics Online, 2022, 18: 11769343221106795. Google Scholar
[19]	邓思雪. 干旱胁迫下不同大豆品种萌发特性及其耐旱性评价[D]. 沈阳: 沈阳农业大学, 2018. Google Scholar
[20]	曹亮, 杜昕, 于高波, 等. 外源褪黑素对干旱胁迫下绥农26大豆鼓粒期叶片碳氮代谢调控的途径分析[J]. 作物学报, 2021, 47(9): 1779-1790. Google Scholar

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干旱胁迫是全球农业生产力的严重制约因素，对农业生产构成持续威胁，干旱的强度和严重程度预计在不久的将来会持续增加. 农业生产中日益严重的缺水和频繁的干旱已经在世界范围内造成巨大的灾害，导致许多作物减产^[1]. 大豆(Glycine max)是重要的油料作物、经济作物及饲料作物，在我国国民经济中占有重要地位. 然而大豆是一个对水分亏缺敏感的作物，在干旱胁迫下，大豆的产量损失可能高达40%，而长期或严重的干旱胁迫甚至会完全破坏种子生产^[2-4]. 因此筛选挖掘大豆抗旱相关基因，解析其在大豆抗/耐旱中的功能和调控网络，将为培育抗/耐旱大豆新种质或新品种提供理论依据和有效基因，具有重要的科学意义和应用价值.

植物抵抗非生物胁迫是一个复杂的协调过程，涉及许多转录调控因子. 在这些转录调节因子中，G-box结合因子(G-box Binding Factor，简称GBF)能够通过识别靶基因启动子中的顺式作用调节元件G-box(CACGTG或CANNTG)，进而调节靶基因的表达^[5]. 已有研究表明G-box结合蛋白参与了多种细胞过程，在植物生长发育和抗/耐生物和非生物逆境方面具有重要作用^[6-8]. B12D是一个G-box结合蛋白，该基因家族在植物发育和应激反应的各个方面中对细胞运输和代谢的调节起着重要作用^[8-11]. B12D家族包含多个成员，B12Dg是该家族成员之一，最早在筛选大麦糊粉层和胚胎的转录组中发现，该基因启动子中含有赤霉素识别元件，在大麦种子发芽时期优势表达^{[8-9, 12]}. 红薯的同源基因在衰老叶片中表达上调^[13]. 拟南芥的B12Dg在种子萌发、植株生长、开花和荚果发育中均有表达^[14]. 地黄(Rehmannia glutinosa)的B12Dg基因受铬、GA和盐胁迫诱导表达^[11]. 小麦B12Dg在干旱胁迫下下调^[15]. 甜菜(Beta vulgaris)B12Dg能够响应盐胁迫^[16]. 多种基因型水稻，在水淹胁迫条件下都能检测到B12Dg基因的诱导表达，而且水淹诱导的基因启动子序列中含有G-box位点^[17]. Almutairi^[18]分析了14个物种中66个B12D蛋白，在预测的B12D启动子中发现了对光、脱落酸、茉莉酸甲酯、细胞分裂素、干旱和热响应的顺式调控元件. 这些研究说明GBF基因在植物响应非生物胁迫中具有重要作用.

为了筛选大豆抗/耐旱相关基因，本研究对大豆幼苗进行了干旱处理并对处理样品进行了转录组分析. Glyma.08G250300基因在干旱处理样品中显著上调，是一个干旱诱导基因(命名为GmDIBL1，Drought Induced B12D Like 1)，可能在大豆抗/耐干旱胁迫中具有作用. 本研究进一步克隆了GmDIBL1基因的基因组序列和互补DNA(complementary DNA，cDNA)序列，分析了其序列特征，检测了该基因在大豆不同组织器官以及不同处理下的表达特性，进而通过构建过表达和基因编辑载体并进行大豆毛状根遗传转化，鉴定了GmDIBL1在大豆耐旱中的功能，以期为深入研究GmDIBL1在大豆抗/耐旱中的生物功能和调控机制奠定基础.

1. 材料与方法

1.1. 植物材料与菌株

试验用大豆品种为‘Williams 82’ ‘晋豆21’和‘绥农26’. 使用的菌株有大肠杆菌菌株感受态细胞DH5α、根癌农杆菌感受态GV3101和发根农杆菌感受态K599.

1.2. 大豆种植及模拟干旱处理

挑选大小和活力一致的‘晋豆21’和‘绥农26’种子于培养箱萌发、生长，生长环境为温度25 ℃，空气相对湿度60%~70%，长日照(昼16 h/夜8 h). 待幼苗的第一对真叶展开后，将大豆幼苗从土壤中轻轻拔出根部，用自来水冲洗根部表面泥土，再用滤纸吸干根部表面水分，用于干旱处理. 干旱处理首先将大豆根部的营养土清洗干净，置于15% PEG6000的1/2MS营养液中进行干旱胁迫. 分别在处理后0 h、6 h、12 h的时间点取植株根样品，做好样品编号. 每个取样时间点取3个生物学重复. 所有样品均用液氮快速冷冻，并在-80 ℃保存备用.

1.3. 植物RNA的提取

挑选大小和活力一致的‘Williams 82’大豆种子于自然条件下萌发、生长，取其根、茎、叶、花、根瘤、荚样品，取3个生物学重复. 使用Trizol方法，提取大豆不同组织以及干旱处理样品的总RNA，并进行琼脂糖凝胶电泳检测.

1.4. 大豆GmDIBL1的扩增

提取得到的RNA样品按照反转录试剂盒(TaKaRa，北京)说明书的具体操作步骤，合成大豆cDNA，-20 ℃保存. 以大豆根cDNA为模板，DIBL1-F/DIBL1-R为引物(DIBL1-F：5'-ttggagaggacagggtaccATGGGGCGTTGGATGAAACCA-3'，DIBL1-R：5'-gccatggatccccgggtaccTTTATCCTCGCTGAAGAAGT-3')，聚合酶链式反应(Polymerase Chain Reaction，简称PCR)扩增得到GmDIBL1.

1.5. 基因的生物信息学分析

以大豆GmDIBL1编码的蛋白为检索序列，利用TBtools软件进行序列同源性比对和相似性搜索. 根据InterPro数据库(https://www.ebi.ac.uk/interpro/result/InterProScan/)预测GmDIBL1编码蛋白的结构域. 根据Panther数据库分析GmDIBL1编码蛋白的蛋白质家族类型. 利用跨膜结构域分析(TMHMM)预测GmDIBL1编码蛋白的跨膜结构域. 根据Phobius预测GmDIBL1编码蛋白的蛋白结构. 选择与大豆GmDIBL1编码蛋白的高度同源序列，利用MEGA11构建进化树.

1.6. 基因表达分析

运用实时荧光定量聚合酶链式反应(Real-time PCR，简称RT-PCR)扩增分析GmDIBL1在不同组织以及干旱处理下的表达水平. 用大豆Actin作内参基因，Actin引物为GmActin-F(5'-CCGGTCGTGACCTCACTGATTTCT-3')和GmActin-R(5'-CATCAGGCAACTCGTAGCTCTTCT-3')，GmDIBL1的引物为GmDIBL1-qRT-F(5'-GGGCGTTGGATGAAACCAGA-3')和GmDIBL1-qRT-R(5'-TCTCTGGTGGTCGAGTACGA-3').

1.7. 过表达载体的构建

选择限制性内切酶KpnⅠ对载体pCAMBIA2300-35s-dsred-eGFP进行酶切，并对酶切后的产物进行胶回收，将GmDIBL1基因片段的回收产物与酶切线性化pCAMBIA2300-35s-dsred-eGFP载体片段的回收产物进行重组反应.

1.8. 基因编辑载体的构建

以pTX2262为模板，KO-DIBL1-F/KO-DIBL1-R为引物，通过PCR得到双靶点目的片段，并使用Golden Gate连接方式与骨架载体pTX2262连接.

1.9. 亚细胞定位检测

将不含终止密码子的编码序列(Coding sequence，简称CDS)片段插入pCAMBIA2300-35S-GFP载体，获得重组质粒35S∷GmDIBL1∷GFP并导入根癌农杆菌GV3101. 将含有重组质粒的农杆菌菌液注射到生长旺盛的本氏烟草叶片中，暗培养过夜后正常光照培养1 d，使用DAPI染料对烟草叶片染色10~20 min，使用激光共聚焦显微镜观察烟草叶片细胞中的荧光信号.

1.10. 大豆毛根转化复合体植株的产生

将大豆种子种入土中大约生长1周龄左右，对大豆幼苗进行农杆菌侵染. 在大豆种子萌发生长期间准备农杆菌材料，将构建好的过表达载体和基因编辑载体以及对应空载体转入发根农杆菌K599的感受态细胞中，并将其涂于含有卡那霉素和链霉素的固体培养基中让其生长1~2 d. 用枪头挑取在上述培养基中生长的农杆菌菌落并活化1 d，吸取活化后的菌液300 μL涂在上述培养基上，于28 ℃恒温培养箱中培养1~2 d后可长成菌膜，平板上的菌膜用刀片刮成菌堆. 将大豆幼苗在子叶下1 cm处沿45°斜切，切口从菌堆中蘸取转入不同质粒的K599菌，将蘸取K599菌的大豆幼苗直接插入蛭石中，放入培养箱中培养10~14 d，培养条件为温度26 ℃，空气相对湿度60%，长日照(昼16 h/夜8 h).

1.11. GmDIBL1大豆毛根转化复合体植株的表型鉴定

两周后，取出植株，在双波长激发光源下检测阳性根，每个植株只留1条健壮的阳性根，其他阳性根和阴性根剔除干净. 同时提取过表达毛状根的RNA进行RT-PCR以确保该基因在根中过量表达，提取基因编辑毛状根的DNA并测序验证以确保该基因被敲除. 使用15%PEG6000处理大豆毛根转化复合体，随时观察其在15% PEG6000处理下的表型. 干旱处理24 h后拍照收集大豆植株表型.

3. 讨论

3.1. GmDIBL1是植物特异的B12D基因的同源基因

在筛选大麦糊粉层和胚的转录组中首次发现了B12D蛋白^[12]. B12D家族蛋白是包含参与膜运输的B12D结构域的跨膜蛋白. 通常情况下，B12D蛋白的长度在86和98 AA之间，包括单个跨膜螺旋. 基序预测和多序列比对显示11种开花植物的B12D蛋白具有较强的保守性^[18]. 本研究中获得的受干旱诱导的B12D同源蛋白GmDIBL1长度为86个氨基酸残基，具有1个跨膜螺旋，含有B12D结构域，与拟南芥、棉花、番茄和玉米的B12D序列高度同源. 这说明GmDIBL1是大豆中B12D的同源基因. 已有的研究报道显示B12D能够响应多种环境胁迫，例如地黄(Rehmannia glutinosa)B12Dg受铬、GA和盐胁迫诱导表达^[11]，小麦B12Dg在干旱胁迫下表达下调^[15]，甜菜(Beta vulgaris)B12Dg能够响应盐胁迫^[16]，5种水稻基因型不同的水稻中B12Dg都能够响应淹水而上调表达^[17]. 生物信息学的分析表明，多数B12D启动子中具有对光、脱落酸、茉莉酸甲酯、细胞分裂素、干旱和热的各种反应元件，少数B12D基因的启动子中存在冷应激、细胞周期、昼夜节律、生长素、水杨酸和赤霉酸的特定元素^[6]. GmDIBL1基因能够响应干旱胁迫而显著上调表达，与其他物种同源基因的诱导表达特征相似. 这在表达响应水平上进一步说明GmDIBL1是B12D的同源基因.

3.2. GmDIBL1在大豆抗/耐干旱胁迫中具有重要作用

现有报道中，尽管对B12D基因的诱导表达特征进行了较多的研究，但对B12D功能的研究较少. 本研究中，由于GmDIBL1基因在根瘤和根中优势表达，利用大豆毛状根体系检测了该基因在抗/耐旱中的功能. 在根中超量表达GmDIBL1基因能够显著提高大豆毛状根复合体的抗旱性，但在敲除该基因的复合体中，抗旱性的降低不明显，这一方面可能是PEG6000的使用浓度过高，没有体现出对照和敲除复合体在抗性方面的差异，另一方面可能是因为大豆是多倍体，存在基因功能冗余的现象，单独敲除一个基因难以在表型上出现差异. 本研究正在创制超量表达GmDIBL1以及敲除多个DIBL1基因的大豆材料，以便深入探索该基因的功能和提供抗旱新种质.

4. 结论

GmDIBL1(Drought Induced B12D Like 1)是一个受干旱诱导的G-box结合蛋白基因，该基因编码86个氨基酸残基，推测分子量为10.1 kD，等电点为9.647. 推导蛋白质序列与其他物种的G-box结合蛋白之间具有较高的保守性，包含B12D结构域和跨膜螺旋，N-端具有信号肽，C-端构成非胞质区域，并定位于细胞核. GmDIBL1基因在根瘤和根中优势表达，在耐旱品种‘晋豆21’根中的表达水平显著高于干旱敏感品种‘绥农26’. 超量表达GmDIBL1基因的毛状根复合体对干旱胁迫的耐受性增强，证明GmDIBL1在大豆抗/耐干旱逆境中具有重要作用. 该研究为深入解析GmDIBL1介导大豆干旱抗性的调控机制奠定了基础.

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Cloning and Functional Identification of Drought Responsive G-box Binding Factor GmDIBL1 in Soybean