Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (19): 4169-4178.doi: 10.3864/j.issn.0578-1752.2021.19.012

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Identification of γ-Glutamyl Transpeptidase AcGGT Gene from Onion (Allium cepa)

XU HuanHuan1,2(),LI Yi1,GAO Wei1,WANG YongQin2(),LIU LeCheng1()   

  1. 1College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, Hubei
    2Vegetable Research Center, Beijing Academy of Agriculture and Forestry/Key Laboratory of Biology and Genetic Improvement of Horticultural (North China), Ministry of Agriculture and Rural Affairs, P.R. China/Beijing Key Laboratory of Vegetable Germplasm Improvement, Beijing 100097
  • Received:2020-11-12 Accepted:2021-01-08 Online:2021-10-01 Published:2021-10-12
  • Contact: YongQin WANG,LeCheng LIU E-mail:sr19951010@126.com;wangyqly@163.com;516119@yangtzeu.edu.cn

RichHTML

35

PDF

934

Abstract:

【Objective】Alliin metabolized by Allium plants had important pharmaceutical value. γ-glutamyl transpeptidase was a key enzyme in the deglutamylation step of the process of alliin synthesis. Studying the function of γ-glutamyl transpeptidase gene in onion can reveal the role of γ-glutamyl transpeptidase in alliin synthesis pathway, providing theoretical basis for alliin synthesis in vitro and laying a foundation for further study on alliin synthesis mechanism. 【Method】Using onion as material, the primers were designed according to onion RNA-seq database, and the gene, γ-glutamyl transpeptidase, was cloned from onion by RT-PCR and analyzed by bioinformatics. The CAMV 35S-AcGGT-GFP vector was used to bombard onion inner epidermis cells with gold powder plasmid microcarrier by particle bombardment technology, and the subcellular localization of AcGGT was determined by fusion green fluorescent expression protein. The Saccharomyces cerevisiae expression vector with AcGGT was constructed, transforming and inducing the expression of AcGGT, and using the method of transforming glutamyl-p-nitroaniline to p-nitroaniline by γ-glutamyl transpeptidase to determine the glutamyl transpeptidase activity of the total protein of Saccharomyces cerevisiae transferred into AcGGT. Real time quantitative PCR was used to analyze the differential expression pattern of the gene in onion tissues. The activity of endogenous transpeptidase in onion tissues was determined by the method of γ-glutamyl transpeptidase catalyzing the production of p-nitroaniline from p-nitroaniline. 【Result】AcGGT was cloned and its length was 1 869 bp. Bioinformatics analysis showed that AcGGT encoded 622 amino acids, protein domain prediction showed that it had glutamyl transpeptidase domain, secondary structure was mainly α - helix, transmembrane region analysis suggested that GGT protein had transmembrane region, amino acid multiple alignment results showed that GGT in plants had certain conservation, evolutionary analysis showed that AcGGT was related to garlic AsGGT2, and the relationship is closest. The fluorescence signal of CaMV 35S-AcGGT-GFP fusion protein was located in the vacuole, indicating that the protein encoded by CaMV 35S-AcGGT-GFP was located in the vacuole. The results of glutamyl transpeptidase activity assay showed that the glutamyl transpeptidase activity of yeast transformed with AcGGT was significantly higher than the control, indicating that the protein encoded by AcGGT had transpeptidase activity. The results of differential expression analysis of AcGGT showed that the expression of AcGGT was mainly in leaf sheath, bulb and leaf sheath followed by. The activity of glutamyl transpeptidase in different tissues, the highest activity in leaf, followed by leaf sheath. Correlation analysis showed that there was no significant correlation between the activity of transglutaminase and the expression of AcGGT. 【Conclusion】The enzymatic reaction data of exogenous AcGGT expression were obtained. The deglutination of alliin synthesis pathway preceded S-oxygenation; there was no significant correlation between AcGGT expression and endogenous transglutaminase activity in onion, suggesting that there may be multiple transglutaminase genes in onion.

Key words: onion, γ-glutamyl transpeptidase, bioinformatics, subcellular localization, eukaryotic expression, expression pattern

Table 1

Primers used in this study"

名称 Name 引物序列 Primer sequence (5′-3′) 引物用途 Use of primer
GGT-F ATGGAACCGGCGCATGATGA 基因克隆
Gene cloning
GGT-R TCACACATGCAGGACTTCCATCT
PYBA1332-GGT-F GGAGAGGACAATTGGAGCTCATGGAACCGGCGCATGATGA 亚细胞定位
Subcellular Localization
PYBA1332-GGT-R CCCTTGCTCACCATGGTACCCACACATGCAGGACTTCCATCT
pYES2-GGT-F GGAATATTAAGCTTGGTACCATGGAACCGGCGCATGATG 真核表达
Prokaryotic Expression
pYES2-GGT-R CCTCTAGATGCATGCTCGAGTCACACATGCAGGACTTCC
AcTUB-F TCAGTCCAGTAGGAGGAATGTCGC 实时荧光定量PCR
qRT-PCR
AcTUB-R CTGTCTTCAGAGGCAAGATGAGCAC
qPCR-GGT-F AGTTGAAGTCACAGAGGCAC 实时荧光定量PCR
qRT-PCR
qPCR-GGT-R CAAGCCTTTCACAGCATCAAG

Fig. 1

PCR amplification of onion AcGGT M：DL5000 DNA Marker;1—3：AcGGT"

Fig. 2

Coding region sequence of AcGGT"

Fig. 3

Sequence alignment of the representative γ-glutamyl transpeptidases sequences from the indicated species Black line: Predicted GGT signal peptide; *: Predicted N-linked glycosylation site; Arrow: Conservative protease cleavage site between large subunit and small subunit"

Fig. 4

Prediction of AcGGT conserved domain and AcGGT secondary structure A: Prediction of the conserved domain of γ-glutamyl transpeptidase; B: Prediction of the secondary structure of γ-glutamyl transpeptidase"

Fig. 5

Phylogenetic tree of AcGGT protein sequences from onion and other species"

Fig. 6

Phylogenetic tree of GGT protein sequences from onion and other species *：Allium cepa AcGGT"

Fig. 7

Subcellular localization of 35S-AcGGT-GFP in onion epidermal cell"

Fig. 8

Activity determination of AcGGT protein heterologously expressed in yeast **: Indicate the significant difference (P<0.01) compared with control"

Fig. 9

Difference of AcGGT expression and activity of transglutaminase in onion R: Root; B: Bulb; S: Sheath; L: Leaf"

[1] JONES M G, HUGHES J, TREGOVA A, MILNE J, TOMSETT A B, COLLIN H A. Biosynthesis of the flavour precursors of onion and garlic. Journal of Experimental Botany, 2004(404):1903-1918.
[2] BEESK N, PERNER H, SCHWARZ D, GEORGE E, KROH L W, ROHN S. Distribution of quercetin-3,4′-O-diglucoside, quercetin-4′- O-monoglucoside, and quercetin in different parts of the onion bulb (Allium cepa) influenced by genotype. Food Chemistry, 2010, 122(3):566-571.
doi: 10.1016/j.foodchem.2010.03.011
[3] 张新茹, 杨晓虹, 王天晓. 葱属植物中甾体皂苷及其药理作用最新研究进展. 解放军药学学报, 2009, 25(2):165-169.
ZHANG X R, YANG X H, WANG T X. Research progress of steroidal saponins and their pharmacological effects in Allium. Pharmaceutical Journal of Chinese People's Liberation Army, 2009, 25(2):165-169. (in Chinese)
[4] 冯长根, 吴悟贤, 刘霞, 李生才. 洋葱的化学成分及药理作用研究进展. 上海中医药杂志, 2003(7):63-65.
FENG C G, WU W X, LIU X, LI S C. Chemical constituents and pharmacological effects of onion. Shanghai Journal of Traditional Chinese Medicine, 2003(7):63-65. (in Chinese)
[5] 弓志青, 靳琼, 陈相艳, 王文亮. 不同品种洋葱粉营养成分分析. 食品科学技术学报, 2014, 32(5):46-49.
GONG Z Q, JIN Q, CHEN X Y, WANG W L. Analysis of nutritional components of different onion powder. Journal of Food Science and Technology, 2014, 32(5):46-49. (in Chinese)
[6] 陈亦辉, 王卫东, 孙月娥. 洋葱中活性物质及生理药理作用研究进展. 中国调味品, 2015, 40(4):129-132+140.
CHEN Y H, WANG W D, SUN Y E. Research progress on active substances and physiological and pharmacological effects of onion. Chinese Condiment, 2015, 40(4):129-132+140. (in Chinese)
[7] NAOKO Y, KAZUKI S. Biosynthesis of S-Alk(en)yl-l-Cysteine Sulfoxides in Allium: Retro Perspective. Sulfur metabolism in higher plants. Fundamental, Environmental and Agricultural Aspects, 2017: 49-60
[8] CONRAD M, SANDIN A, FORSTER H, SEILER A, FRIJHOFF J, DAGNELL M, BORNKAMM G W, RÅDMARK O, HOOFT VAN HUIJSDUIJNEN R, ASPENSTRÖM P, BÖHMER F, OSTMAN A. 12/15-lipoxygenase-derived lipid peroxides control receptor tyrosine kinase signaling through oxidation of protein tyrosine phosphatases. Proceedings of the National Academy of sciences of the USA, 2010, 107(36):15774-15779.
[9] TIAN S Y, HAO C C, XU G K, YANG J J, SUN R G. Optimization conditions for extracting polysaccharide from Angelica sinensis and its antioxidant activities. Journal of Food and Drug Analysis, 2017, 25(4):766-775
doi: 10.1016/j.jfda.2016.08.012
[10] ORLOWSKI M, MEISTER A. Gamma-glutamyl-p-nitroanilide: A new convenient substrate for determination and study of l- and d-gamma-glutamyltranspeptidase activities. Biochimica et Biophysica Acta- Proteins and Proteomics, 1963, 73(4):679-681.
[11] NAOAKI M, NAGATOSHI I, NORBERT W. Aged garlic extract inhibits CD36 expression in human macrophages via modulation of the PPARgamma pathway. Phytotherapy Research, 2010, 24(4):602-608.
doi: 10.1002/ptr.3008
[12] 王辉, 李景明, 马钊. 洋葱中含硫化合物的生理功效. 食品工业科技, 2005, 26(5):187-189.
WANG H, LI J M, MA Z. Physiological effects of sulfur compounds in onion. Food Industry Science and Technology, 2005, 26(5):187-189. (in Chinese)
[13] YOSHIMOTO N, YABE A, SUGINO Y. Garlic γ-glutamyl transpeptidases that catalyze deglutamylation of biosynthetic intermediate of alliin. Frontiers in Plant Science, 2014, 5:758.
[14] PETROPOULOS S, DI GIOIA F, NTATSI G. Vegetable organosulfur compounds and their health promoting effects. Current Pharmaceutical Design, 2017, 23(19):2850-2875.
doi: 10.2174/1381612823666170111100531
[15] HUGHES J, TREGOVA A, TOMSETT A B, JONES M G, COSSTICK R, COLLIN H A. Synthesis of the flavour precursor, alliin, in garlic tissue cultures. Phytochemistry, 2005, 66(2):187-194.
doi: 10.1016/j.phytochem.2004.11.009
[16] YU T H, WU C M, HO C T. Meat-like flavor generated from thermal interactions of glucose and alliin or deoxy-alliin. Journal of Agricultural and Food Chemistry, 1994, 42(4):1005-1009.
doi: 10.1021/jf00040a032
[17] LANZOTTI V, SCALA F, BONANOMI G. Compounds from Allium species with cytotoxic and antimicrobial activity. Phytochemistry Reviews, 2014, 13(4):769-791.
doi: 10.1007/s11101-014-9366-0
[18] SHUAI Y, ZHANG T, MU W, JIANG B. Purification and Characterization of γ-Glutamyl transpeptidase from Bacillus subtilis SK11.004. Journal of Agricultural and Food Chemistry, 2011, 59(11):6233-6238.
doi: 10.1021/jf2003249
[19] MELINDA N M, JANET P S. Purified γ-glutamyl transpeptidases from tomato exhibit high affinity for glutathione and glutathione S-conjugates. Plant Physiology, 2000, 122(4):1417-1426.
doi: 10.1104/pp.122.4.1417
[20] LI J, HUANG J, YIN J, WU N, SONG J, ZHANG L, JIANG T. Rapid purification and characterization of γ‐glutamyl‐transpeptidase from shiitake mushroom (Lentinus edodes). Journal of Food Science, 2012, 77(4/6):C640-C645.
doi: 10.1111/j.1750-3841.2012.02725.x
[21] FUJII T, MATSUTOMO T, KODERA Y. Changes of S- Allylmercaptocysteine and γ-Glutamyl- S-allylmercaptocysteine contents and their putative production mechanisms in garlic extract during the aging process. Journal of Agricultural and Food Chemistry, 2018, 66(40):10506-10512.
doi: 10.1021/acs.jafc.8b02541
[22] URS W, SHAW M L, LANCASTER J E. Effect of freezing upon alliinase activity in onion extracts and pure enzyme preparations. Journal of the Science of Food and Agriculture, 1994, 64(3):315-318.
doi: 10.1002/(ISSN)1097-0010
[23] SUN Y, HU J, WANG W, ZHANG B, SHEN Y. Characterization of γ-lutamyl transpeptidases from dormant garlic and onion bulbs. Food Science and Nutrition, 2019, 7(2):499-505.
doi: 10.1002/fsn3.2019.7.issue-2
[24] SHAW M L, PITHER-JOYCE M D, MCCALLUM J A. Purification and cloning of a gamma-glutamyl transpeptidase from onion (Allium cepa). Phytochemistry, 2005, 66(5):515-522.
doi: 10.1016/j.phytochem.2005.01.017
[25] BRADFORD M. A rapid and sensitive method for the quantitation of microgam quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, 72(1/2):248-254.
doi: 10.1016/0003-2697(76)90527-3
[26] SUZUKI T, SUGII M, KAKIMOTO T. Metabolic incorporation of L-valine-[14C] into S-(2-carboxypropyl) glutathione and S-(2- carboxypropyl) cysteine in garlic. Chemical and Pharmaceutical Bulletin, 1962, 10(4):328-331.
doi: 10.1248/cpb.10.328
[27] BAYAN L, KOULIVAND P H, GORJI A. Garlic: A review of potential therapeutic effects. Avicenna Journal of Phytomedicine, 2014, 4(1):1-14.
[28] TURNBULL A, GALPIN I J, COLLIN H A, SMITH J L. Comparison of the onion plant (Allium cepa) and onion tissue culture: III. Feeding of 14C labeled precursors of the flavor precursor compounds. New Phytologist, 2006, 85(4):483-487.
doi: 10.1111/nph.1980.85.issue-4
[29] LANCASTER J E, SHAW M L. γ-Glutamyl peptides in the biosynthesis of S-alk(en)yl-l-cysteine sulphoxides (flavour precursors) in Allium. Phytochemistry, 1989, 28(2):455-460.
doi: 10.1016/0031-9422(89)80031-7
[30] DONG Y, LISK D, BLOCK E, IP C. Characterization of the bi-ological activity of gamma- glutamyl- Se- methylselenocysteine: A novel, naturally occurring anticancer agent from garlic. Cancer Research, 2001, 61(7):2923-2928.
[31] LANCASTER J E, AND M L S, RANDLE W M. Differential hydrolysis of alk(en)yl cysteine sulphoxides by alliinase in onion macerates: Flavour implications. Journal of the Science of Food and Agriculture, 1998, 78(3):367-372
doi: 10.1002/(ISSN)1097-0010
[32] SUN X, ZHU S, LI N. A chromosome-level genome assembly of garlic (Allium sativum L.) provides insights into genome evolution and allicin biosynthesis. Molecular Plant, 2020, 13(9):1328-1339.
doi: 10.1016/j.molp.2020.07.019
[1] LI ShiJia,LÜ ZiJing,ZHAO Jin. Identification of R2R3-MYB Subfamily in Chinese Jujube and Their Expression Pattern During the Fruit Development [J]. Scientia Agricultura Sinica, 2022, 55(6): 1199-1212.
[2] GUO ShaoLei,XU JianLan,WANG XiaoJun,SU ZiWen,ZHANG BinBin,MA RuiJuan,YU MingLiang. Genome-Wide Identification and Expression Analysis of XTH Gene Family in Peach Fruit During Storage [J]. Scientia Agricultura Sinica, 2022, 55(23): 4702-4716.
[3] ZHANG Rui,ZHANG XueYao,ZHAO XiaoMing,MA EnBo,ZHANG JianZhen. Antibody Preparation and Subcellular Localization of LmKnk3-5′ in Locusta migratoria [J]. Scientia Agricultura Sinica, 2022, 55(2): 329-338.
[4] CHEN FengQiong, CHEN QiuSen, LIN JiaXin, WANG YaTing, LIU HanLin, LIANG BingRuoShi, DENG YiRu, REN ChunYuan, ZHANG YuXian, YANG FengJun, YU GaoBo, WEI JinPeng, WANG MengXue. Genome-Wide Identification of DIR Family Genes in Tomato and Response to Abiotic Stress [J]. Scientia Agricultura Sinica, 2022, 55(19): 3807-3821.
[5] ZHANG YunXiu,JIANG Xu,WEI ChunXue,JIANG XueQian,LU DongYu,LONG RuiCai,YANG QingChuan,WANG Zhen,KANG JunMei. The Functional Analysis of High Mobility Group MsHMG-Y Involved in Flowering Regulation in Medicago sativa L. [J]. Scientia Agricultura Sinica, 2022, 55(16): 3082-3092.
[6] YAN DuoZi,CAI Ni,WANG Feng,NONG XiangQun,WANG GuangJun,TU XiongBing,ZHANG ZeHua. Expression in vitro of Metarhizium anisopliae Adhesin MAD1 and Its Effect on Inducing Response in Peanut [J]. Scientia Agricultura Sinica, 2021, 54(4): 744-753.
[7] WANG Yong,LI SiYan,HE SiRui,ZHANG Di,LIAN Shuai,WANG JianFa,WU Rui. Prediction and Bioinformatics Analysis of BLV-miRNA Transboundary Regulation of Human Target Genes [J]. Scientia Agricultura Sinica, 2021, 54(3): 662-674.
[8] GE XinZhu,SHI YuXing,WANG ShaSha,LIU ZhiHui,CAI WenJie,ZHOU Min,WANG ShiGui,TANG Bin. Sequence Analysis of Harmonia axyridis Pyruvate Kinase Gene and Its Regulation of Trehalose Metabolism [J]. Scientia Agricultura Sinica, 2021, 54(23): 5021-5031.
[9] WANG Hao,YIN Lian,LIU JieXia,JIA LiLi,DING Xu,SHEN Di,FENG Kai,XU ZhiSheng,XIONG AiSheng. The Carotenoid Cleavage Dioxygenases Gene AgCCD4 Regulates the Pigmentation of Celery Tissues with Different Colors [J]. Scientia Agricultura Sinica, 2021, 54(15): 3279-3294.
[10] SHI GuoLiang,WU Qiang,YANG NianWan,HUANG Cong,LIU WanXue,QIAN WanQiang,WAN FangHao. Gene Cloning, Expression Pattern and Molecular Characterization of Chitin Deacetylase 2 in Cydia pomonella [J]. Scientia Agricultura Sinica, 2021, 54(10): 2105-2117.
[11] SUN HongYing,WANG Yan,LI WeiJia,ZHU TianShu,JIANG Ying,XU Yan,WU QingYue,ZHANG ZhiHong. Expression Characteristics and Function of FveD27 in Woodland Strawberry [J]. Scientia Agricultura Sinica, 2021, 54(10): 2179-2191.
[12] ZHANG YuFei,CAO ManYuan,WANG LiYing,ZHAO WeiGang,LI XiaoXia,CHANG Tong,XU BaoZeng. Eukaryotic Expression, Purification and Biological Activity of Recombinant Cervus Nippon Activin A Protein [J]. Scientia Agricultura Sinica, 2020, 53(5): 1058-1070.
[13] XING QiKai,LI LingXian,CAO Yang,ZHANG Wei,PENG JunBo,YAN JiYe,LI XingHong. Prediction and Analysis of Candidate Secreted Proteins from the Genome of Lasiodiplodia theobromae [J]. Scientia Agricultura Sinica, 2020, 53(24): 5027-5038.
[14] YUAN XinBo,CHENG TingTing,XI XiaoHan,CHEN ZhangYu,WANG RuiHong,KE WeiDong,GUO HongBo. Screening of Polyphenol Oxidase Interaction Proteins from Nelumbo nucifera and Their Verification [J]. Scientia Agricultura Sinica, 2020, 53(18): 3777-3791.
[15] HAO ShuLin,CHEN HongWei,LIAO FangLi,LI Li,LIU ChangYan,LIU LiangJun,WAN ZhengHuang,SHA AiHua. Analysis of F-Box Gene Family Based on Salt-Stressed Transcriptome Sequencing in Vicia faba L. [J]. Scientia Agricultura Sinica, 2020, 53(17): 3443-3454.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd