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作物学报 ›› 2021, Vol. 47 ›› Issue (4): 761-769.doi: 10.3724/SP.J.1006.2021.01046

• 研究简报 • 上一篇    下一篇

小麦谷氨酰胺合成酶同工酶转录特点及其启动子序列分析

王小纯1,2(), 王露露1, 张志勇1, 秦步坛1, 于美琴2, 韦一昊1, 马新明1,*()   

  1. 1河南粮食作物协同创新中心 / 河南农业大学, 河南郑州 450002
    2河南农业大学生命科学学院, 河南郑州450002
  • 收稿日期:2020-06-03 接受日期:2020-10-17 出版日期:2021-04-12 网络出版日期:2020-11-02
  • 通讯作者: 马新明
  • 作者简介:E-mail: xiaochun.w@163.com
  • 基金资助:
    国家重点研发计划项目(2016YFD0300205);河南省现代农业产业技术体系项目(S2010-01-G04)

Transcription characteristics of wheat glutamine synthetase isoforms and the sequence analysis of their promoters

WANG Xiao-Chun1,2(), WANG Lu-Lu1, ZHANG Zhi-Yong1, QIN Bu-Tan1, YU Mei-Qin2, WEI Yi-Hao1, MA Xin-Ming1,*()   

  1. 1Collaborative Innovation Center of Henan Grain Crops, Henan Agricultural University, Zhengzhou 450002, Henan, China
    2College of Life Science, Henan Agricultural University, Zhengzhou 450002, Henan, China
  • Received:2020-06-03 Accepted:2020-10-17 Published:2021-04-12 Published online:2020-11-02
  • Contact: MA Xin-Ming
  • Supported by:
    National Key Research and Development Program of China(2016YFD0300205);Modern Agricultural Technology System in Henan Province(S2010-01-G04)

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摘要：

谷氨酰胺合成酶是小麦氮同化关键酶, 分为胞液型和质体型(TaGS2)两类, 其中胞液型TaGS又分为TaGS1、TaGSr和TaGSe。为了研究异源六倍体小麦A、B、D染色体组TaGS同工酶表达差异及调控机制, 本项目利用三代测序技术测定了TaGS同工酶基因转录水平, 依据中国春基因组序列克隆了豫麦49的12个TaGS同工酶启动子, 并对其序列进行了分析。结果表明, TaGS1主要由6B染色体基因转录, TaGSe和TaGSr主要由4D染色体基因转录, TaGS2主要由2D染色体基因转录, 不同TaGS同工酶转录起始位点距起始密码子ATG的距离不同。启动子元件分析显示, 6B染色体上的TaGS1启动子有较多W-box、AC-I、ABRE、as-1和茉莉酸甲酯等响应元件, 4D染色体上的TaGSe启动子有较多胁迫响应转录因子(MYB、MBS、LTR等)结合元件和植物生长素响应元件, 4D染色体上的TaGSr启动子有较多WRE3等转录因子结合元件, 2D染色体上的TaGS2启动子有较多A-box、WRE3、ARE及AT富集区。不同TaGS同工酶启动子顺式元件种类、数目及排列顺序均不同, 为进一步研究GS同工酶调控机制奠定了基础。

关键词: 小麦, GS同工酶, 转录, 启动子, 顺式作用元件

Abstract:

As a key enzyme for nitrogen assimilation in wheat, glutamine synthetase is grouped into two classes: cytosolic GS and chloroplastic GS (TaGS2), and cytosolic GS includes TaGS1, TaGSr, and TaGSe. In order to study the expression characteristics and regulatory mechanisms of GS isozymes in chromosome A, B, and D of heterohexaploid wheat, transcripts of TaGS isoforms were analyzed based on the third-generation sequencing technology transcriptome analysis, and 12 promoters of TaGS isozymes of Yumai 49 were cloned based on Chinese Spring genome, and the sequence of the promoters were analyzed. The results showed that TaGS1 was mainly transcribed on chromosome 6B, TaGSe and TaGSr on chromosome 4D, and TaGS2 on chromosome 2D. Furthermore, the distance from initiation codon ATG to initiation site of transcript for each promoter of TaGS was distinct. Promoter element analysis showed that the promoter of TaGS1 in 6B had more W-box, AC-I, ABRE, as-1, and methyl jasmonic response elements, the promoter of TaGSe in 4D had more stress response elements (MYB, MBS, LTR, etc.) and auxin response element, the promoter of TaGSr in 4D had more WRE3 and transcript factor response elements, the promoter of TaGS2 in 2D had more A-box, WRE3, ARE, and an AT enrichment region. In summary, the number, type and order of cis-elements of different promoters of TaGS isozymes were distinct, which provided the foundation for further study on the regulation mechanism of TaGS isozymes.

Key words: wheat, GS isoforms, transcription, promoter, cis-element

图1

不同染色体上TaGS同工酶基因表达特点"

表1

TaGS同工酶启动子扩增引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 退火温度
Annealing temperature (℃)		 PCR长度
PCR fragment (bp)		 5′端非编码区 5′NCS (bp)
GS1-6AL S CACTGCCTTCTCAGGCTTGTTA 58 2000 1594
GS1-6AL A GCCCCATGAATTTAGCATCG
GS1-6BL S TCTTAGTCCAACGTGGCATCATAGG 62 2033 1548
GS1-6BL A AGACGCCAAACTACGACTACG
GS1-6DL S CACATTCACACGTGGTTTCCTCT 58 2261 1470
GS1-6DL A GCAAAAACCTCTTCTCGTCGC
GSe-4AL S TCACCCATAATCATGCTGTGCAAAT 63 2538 1557
GSe-4AL A TACCATATGTACTCGGCGATGATCTTG
GSe-4BL A ATGGGAGGTTGGGACAGTGC 58 2508 1565
GSe-4BL S GATGCGAAGCATACGCGC
GSe-4DS S TCTGAAGATGCTCATGAGTTGCTCAAT 62 2536 1557
GSe-4DS A TACCATATGTACTCGGCGATGATCTTG
GSr-4AS S GTAATGTGGCTACGGTGTGAGTCTGTT 61 2155 1548
GSr-4AS A GGGTGCAGAATGCAAGTAGC
GSr-4BL S TCATCTCCGTGAGGAGGTCTTGA 62 2308 1615
GSr-4BL A TACTCGACGATGATCTTGTCGGTG
GSr-4DL S GCCCGTTGTACCACTGTTATCGAT 62 1624 1467
GSr-4DL A TACTCGACGATGATCTTGTCGGTG
GS2-2AL S GCATTCCTGGCTTGCATGTCTAGA 58 2008 1499
GS2-2AL A ATGATCTTGTCGGTGAAGGGC
GS2-2BL S ATTTCGCCGAGTTCAGGTCGAG 63 2153 1419
GS2-2BL A ATGATCTTGTCGGTGAAGGGC
GS2-2DL S CAGCGATCCTAGCCTTCGAAAAG 60 1878 1097
GS2-2DL A ATGATCTTGTCGGTGAAGGGC

图2

不同染色体TaGS同工酶启动子PCR扩增产物鉴定 箭头所指为目的片段。M: DNA分子量标记。"

图3

TaGS同工酶启动子质粒PCR鉴定箭头所指为目的片段。M: DNA分子量标记。The arrows are the pointed to the target segment. M: marker."

图4

不同TaGS同工酶启动子顺式元件种类及分布 不同颜色代表不同类型的响应元件, 颜色和形状代表一个具体的响应元件。红色: 激素响应元件; 绿色: 功能未知的响应元件; 蓝色: 基础元件; 黑色: 转录因子结合位点; 紫色: 胁迫响应元件; 粉色: 组织特异性响应元件; 黄色: 光响应元件。"

图5

不同TaGS同工酶转录起始位点预测 TATA-box为RNA聚合酶II识别位点。"

[1] Miflin B J, Habash D Z. The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. J Exp Bot, 2002,53:979-987.
pmid: 11912240
[2] Ortega J L, Wilson O L, Sengupta-Gopalan C. The 5′ untranslated region of the soybean cytosolic glutamine synthetase β1 gene contains prokaryotic translation initiation signals and acts as a translational enhancer in plants. Mol Genet Genom, 2012,287:881-893.
[3] Keys A J. The re-assimilation of ammonia produced by photorespiration and the nitrogen economy of C3 higher plants. Photosynth Res, 2006,87:165-175.
[4] Konishi N, Saito M, Imagawa F, Kanno K, Yamaya T, Kojima S. Cytosolic glutamine synthetase isozymes play redundant roles in ammonium assimilation under low-ammonium conditions in roots of Arabidopsis thaliana. Plant Cell Physiol, 2018,59:601-613.
[5] Kichey T, Heumez E, Pocholle D, Pageau K, Vanacker H, Dubois F, Gouis J L, Hirel B. Combined agronomic and physiological aspects of nitrogen management in wheat highlight a central role for glutamine synthetase. New Phytol, 2006,169:265-278.
pmid: 16411930
[6] Li H M, Liang H, Li Z, Li Z, Tang Z X, Fu S L, Geng Y Y, Yan B J, Ren Z L. Dynamic QTL analysis of protein content and glutamine synthetase activity in re-combinant inbred wheat lines. Genet Mol Res, 2015,14:8706-8715.
[7] Kichey T, Hirel B, Heumez E, Dubois F, Le Gouis J. In winter wheat ( Triticum aestivum L.) post-anthesis nitrogen uptake and remobilization to the grain correlates with agronomic traits and nitrogen physiological markers. Field Crops Res, 2007,102:22-32.
[8] Bernard S M, Moller A L, Dionisio G, Kichey T, Jahn T P, Dubois F, Baudo M, Lopes M S, Tercé-Laforgue T, Foyer C H, Parry M A, Forde B G, Araus J L, Hirel B, Schjoerring J K, Habash D Z. Gene expression, cellular localization and function of glutamine synthetase isozymes in wheat ( Triticum aestivum L.). Plant Mol Biol, 2008,67:89-105.
pmid: 18288574
[9] 王小燕, 于振文. 不同小麦品种主要品质性状及相关酶活性研究. 中国农业科学, 2005,38:1980-1988.
Wang X Y, Yu Z W. Study on main quality traits and related enzyme activities of different wheat varieties. Sci Agric Sin, 2005,38:1980-1988 (in Chinese with English abstract).
[10] Cai H, Zhou Y, Xiao J, Li X, Zhang Q, Lian X. Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice. Plant Cell Rep, 2009,28:527-537.
[11] 贾喜婷, 韦一昊, 谷明鑫, 石爱博, 王小纯. 过表达TaGS1/TaGS2对烟草抗盐能力的影响及其机制. 中国烟草学报, 2017,121:112-117.
Jia X T, Wei Y H, Gu M X, Shi A B, Wang C H. Effects of overexpression of TaGS1/TaGS2 on tobacco salt resistance and its mechanism. Chin J Tob Sci, 2017,121:112-117 (in Chinese with English abstract).
[12] Hoshida H, Tanaka Y, Hibino T, Hayashi Y, Tanaka A, Takabe T, Takabe T. Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase. Plant Mol Biol, 2000,43:103-111.
[13] 张同勋. 小麦谷氨酰胺合成酶在氮素代谢中的功能分析. 河南农业大学硕士学位论文, 河南郑州, 2012. pp 7-32.
Zhang T X. Functional Analysis of Wheat Glutamine Synthase in Nitrogen Metabolism. MS Thesis of Henan Agricultural University, Zhengzhou, Henan, China, 2012. pp 7-32 (in Chinese with English abstract).
[14] Céline M D, Michèle R C, Karine P, Maud L, Olivier G, Joceline K, Marie H V, Magali F, Tiphanie J, Akira S. Glutamine synthetase-glutamate synthase pathway and Glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco. Plant Physiol, 2006,140:444-456.
[15] Moison M, Marmagne A, Dinant S, Soulay F, Azzopardi M, Lothier J, Citerne S, Morin H, Legay N, Chardon F, Avice J C, Reisdorf-Cren M, Masclaux-Daubresse C. Three cytosolic glutamine synthetase isoforms located in different order veins work together for N remobilization and seed filling in Arabidopsis. J Exp Bot, 2018,69:4379-4393.
[16] Tabuchi M, Sugiyama K, Ishiyama K, Inoue E, Sato T, Takahashi H, Yamaya T. Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1:1, a cytosolic glutamine synthetase1:1. Plant J, 2005,42:641-651.
[17] Tabuchi M, Abiko T, Yamaya T. Assimilation of ammonium ions and reutilization of nitrogen in rice (Oryza sativa L.). J Exp Bot, 2007,58:2319-2327.
[18] Gómez-Maldonado J, Avila C, Torre F, Cañas R, Cánovas F M, Campbell M M. Functional interactions between a glutamine synthetase promoter and MYB proteins. Plant J, 2004,39:513-526.
[19] 董佳敏, 徐永清, 彭丽娜, 冯旭, 姚树宽, 赵巧芩, 李凤兰, 胡宝忠. 小麦品种东农冬麦2号根中TaEXPA7部分同源基因的克隆及表达特性分析. 麦类作物学报, 2017,37(11):25-33.
Dong J M, Xu Y Q, Peng L N, Feng X, Yao S K, Zhao Q C, Li L F, Hu B Z. Cloning and expression characteristics analysis of some homologous genes TaEXPA7 in root of winter wheat Dongnong No.2 variety. J Wheat Crops, 2017,37(11):25-33 (in Chinese with English abstract).
[20] 何辉. 光皮桦BlOFPs基因克隆及其互作蛋白分析. 浙江农林大学硕士学位论文, 浙江杭州, 2016. pp 15-76.
He H. Cloning of the BlOFPs Gene and Analysis of Its Interaction Proteins in Birch. MS Thesis of Zhejiang A & F University, Hangzhou, Zhejiang, China, 2016. pp 15-76 (in Chinese with English abstract).
[21] 李志英, 牟红珍, 高丁梅, 丁国平, 马婷, 王盛. 本氏烟I型启动子的克隆及其转录起始位点分析. 中国生物工程杂志, 2014,34(1):28-35.
Li Z Y, Mou H Z, Gao D M, Ding G P, Ma T, Wang S. Cloning of cigarette type I promoter and analysis of its transcriptional starting sites. Chin J Biol Eng, 2014,34(1):28-35 (in Chinese with English abstract).
[22] 李一琨, 王金发. 高等植物启动子研究进展. 植物学通报, 1998,15(增刊1):1-6.
Li Y K, Wang J F. Research progress of higher plant promoters. Bot Bull, 1998,15(S1):1-6 (in Chinese with English abstract).
[23] 路静, 赵华燕, 何奕昆, 宋艳茹. 高等植物启动子及其应用研究进展. 自然科学进展, 2004,14:856-862.
Lu J, Zhao H Y, He Y K, Song Y R. Advances in higher plant promoters and their applications. Adv Nat Sci, 2004,14:856-862 (in Chinese with English abstract).
[24] 王昊龙, 韩俊杰, 李卫华, 刘伟. 抗性淀粉含量不同的小麦品种(系)淀粉分支酶EIIn和EII6基因多态性分析. 新疆农业科学, 2015,52:981-987.
Wang H L, Han J J, Li W H, Liu W. Resistant starch content of different wheat varieties (or lines) of starch branching enzyme EIIn and EII6 gene polymorphism analysis. Xinjiang Agric Sci, 2015,52:981-987 (in Chinese with English abstract).
[25] 扆珩, 李昂, 刘惠民, 景蕊莲. 小麦蛋白磷酸酶2A基因TaPP2AbB″-α启动子的克隆及表达分析. 作物学报, 2016,42:1282-1290.
Yi H, Li A, Liu H M, Jing R L. Wheat protein phosphatase 2A gene TaPP2AbB"-α promoter cloning and expression analysis. Acta Agron Sin, 2016,42:1282-1290 (in Chinese with English abstract).
[26] 邵宇鹏, 杨明明, 包格格, 孙英楠, 杨强, 李文滨, 王志坤. 大豆GmWRI1a基因启动子克隆及其功能分析. 中国油料作物学报, 2019,41:517-523.
Shao Y P, Yang M M, Bao G G, Sun Y N, Yang Q, Li W B, Wang Z K. Cloning and functional analysis of soybean GmWRI1a promoter. Chin J Oil Crop Sci, 2019,41:517-523 (in Chinese with English abstract).
[27] 魏文杰, 邓霞, 杨淑慎. 非生物胁迫下小麦TaGAPCp1基因启动子的功能分析. 福建师范大学学报(自然科学版), 2019,35(2):76-84.
Wei W J, Deng X, Yang S S. Functional analysis of TaGAPCp1 gene promoter in wheat under abiotic stress. J Fujian Norm Univ(Nat Sci Edn), 2019,35(2):76-84 (in Chinese with English abstract).
[28] 常建忠, 董春林, 张正, 乔麟轶, 杨睿, 蒋丹, 张彦琴, 杨丽莉, 吴佳洁, 景蕊莲. 小麦抗逆相关基因TaSAP1的5′非翻译区含子功能分析. 作物学报, 2019,45:1311-1318.
Chang J Z, Dong C L, Zhang Z, Qiao L Y, Yang R, Jiang D, Zhang Y Q, Yang L L, Wu J J, Jing R L. Functional analysis of 5' non-translational subdomains of stress-related gene TaSAP1 in wheat. Acta Agron Sin, 2019,45:1311-1318 (in Chinese with English abstract).
[29] 金正勋, 李丹, 李明月, 同拉嘎, 潘冬, 张玉磊, 王海微, 韩云飞, 张忠臣. 水稻谷氨酰胺合成酶同工型基因转录表达量与启动子结构关系分析. 东北农业大学学报, 2017,48(10):1-10.
Jin Z X, Li D, Li M Y, Tong L G, Pan D, Zhang Y L, Wang H W, Han Y F, Zhang Z C. Relationship between transcriptional expression of glutamine synthase isotype gene and promoter structure in rice. J Northeast Agric Univ, 2017,48(10):1-10 (in Chinese with English abstract).
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