Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (17): 3348-3358.doi: 10.3864/j.issn.0578-1752.2014.17.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES • Previous Articles     Next Articles

Cloning of Glutamine Synthetase BnGS2 Allele Genes from Ramie (Boehmeria nivea L.) and Study on Gene-Transforming Tobacco

ZHENG Jian-shu, YU Chun-ming, CHEN Ping, WANG Yan-zhou, TAN Long-tao, CHEN Ji-kang, ZHU Tao-tao, LU Ling-xiao, ZHU Juan-juan, DUAN Ye-hui, XIONG He-ping   

  1. Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205
  • Received:2014-03-03 Online:2014-09-01 Published:2014-05-26

RichHTML

1

PDF

795

Cited

1

Abstract: 【Objective】 The objective of this paper is to clone and construct the over-expression vector of two glutamine synthetase (BnGS2) allele genes to study their effects on nitrogen metabolism of transgenic tobacco plants. 【Method】 The ramie transcriptome unigenes and RT-PCR were used to isolate BnGS2 allele genes which further identified by TaqⅠdigestion in self-bred F1 and parents. Sequence and structure were analyzed by bioinformatics. In addition, BnGS2 allele genes over-expression vector was constructed respectively according to homologous recombination technology and transformed through Agrobacterium tumefaciens LBA4404 using “leaf-disk” transformation method into Nicotiana tabacum. The transgenic T0 plants were verified by Kan screening and DNA PCR determination. The qRT-PCR was used for determining the relative expression levels of BnGS2 allele genes in T1 transgenic tobacco plants as well as the leaf GS activity, fresh weight, plant height, leaf soluble protein and total nitrogen content of transgenic plants were determined. 【Result】 Two BnGS2 allele genes with length of 1 340 bp containing 1 293 bp ORF region encoded polypeptide of 430 amino acids were isolated for the first time from ramie, named BnGS2-1 and BnGS2-2. The diversity of nucleotide in 11 sites among BnGS2 allele genes resulted in amino acid residues substitution at sites 195 and 382 (Pro-195 and Asp-382 in BnGS2-1, Thr-195 and Ser-382 in BnGS2-2). The NCBI Blastp analysis displayed that BnGS2 was close to Pisum sativum, Vigna radiate, Glycine max, Phaseolus vulgaris, and Medicago truncatula. In addition, the over-expression vectors of BnGS2-1 and BnGS2-2 were successfully constructed according to homologous recombination technology and the independent transgenic plants over-expressing BnGS2-1 and BnGS2-2 were obtained, respectively. Compared with wild type plants, the transgenic plants exhibited significant increase in leaf GS activity, fresh weight and leaf soluble protein content. The plant height and leaf total nitrogen content were slightly higher but not reached significant levels. However, these parameters between the transgenic plants with over-expression different BnGS2 allele gene (BnGS2-1 or BnGS2-2) did not exhibit significant difference. 【Conclusion】 Over-expression BnGS2 allele genes (BnGS2-1 and BnGS2-2) in the transgenic plants respectively resulted in a higher biomass and enhanced nitrogen use efficiency. In addition, there was no obvious difference in function between isolated BnGS2-1 and BnGS2-2.

Key words: Boehmeria nivea L. , GS2 allele genes cloning , over-expression , transgenic , nitrogen use efficiency

[1]Tingey S V, Tsai F Y, Edwards J W, Walker E L, Coruzzi G M. Chloroplast and cytosolic glutamine synthetase are encoded by homologous nuclear genes which are differentially expressed in vivo. Biology Chemistry, 1988, 263(20): 9651-9657.
[2]朱爱国. 苎麻主要品质性状的研究[D]. 北京: 中国农业科学院, 2001.
Zhu A G. Studies on main quality characters of ramie[D]. Beijing: Chinese Academy of Agricultural Science, 2001. (in Chinese)
[3]熊和平. 苎麻多功能开发潜力及利用途径. 中国麻作, 2001, 23(1): 22-25.
Xiong H P. The potential of ramie multi-functional development and utilization. Plant Fiber Science in China, 2001, 23(1): 22-25. (in Chinese)
[4]揭雨成, 康万利, 邢虎成, 佘玮. 苎麻饲用资源筛选. 草业科学, 2009, 26(9): 30-33.
Jie Y C, Kang W L, Xing H C, She W. Screening of ramie gene resources for forage. Pratacultural Science, 2009, 26(9): 30-33. (in Chinese)
[5]Cren M, Hirel B. Glutamine synthetase in higher plants: Regulation of gene and protein expression from the organ to the cell. Plant and Cell Physiology, 1999, 40: 1187-1193.
[6]Goodall A J, Kumar P, Tobin A K. Identification and expression analyses of cytosolic glutamine synthetase genes in barley (Hordeum vulgare L.). Plant and Cell Physiology, 2013, 54(4): 492-505.
[7]Martin A, Lee J, Kichey T, Gerentes D, Zivy M, Tatout C, Dubois F, Balliau T, Valot B, Davanture M, Terce-Laforgue T, Quillere I, Coque M, Gallais A, Gonzalez-Moro M, Bethencourt L, Habash D Z, Lea P J, Charcosset A, Perez P, Murigneux A, Sakakibara H, Edwards K J, Hirel B. Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production. The Plant Cell, 2006, 18: 3252-3274.
[8]Yamaya T, Obara M, Nakajima H, Sasaki S, Hayakawa T, Sato     T. Genetic manipulation and quantitative-trait loci mapping for nitrogen recycling in rice. Journal of Experimental Botany, 2002, 53: 917-925.
[9]Becker T W, Caboche M, Carrayol E, Hirel B. Nucleotide sequence of a tobacco cDNA encoding plastidic glutamine synthetase and light inducibility, organ specificity and diurnal rhythmicity in the expression of the corresponding genes of tobacco and tomato. Plant Molecular Biology, 1992, 19: 367-379.
[10]Galais A, Hirel B. An approach to the genetics of nitrogen use efficiency in maize. Journal of Experimental Botany, 2004, 55: 295-306.
[11]Silveira J A G, Viegas R A, Rocha I M A, Moreira A C O M, Moreira R A, Oliveira J T A. Proline accumulation and glutamine synthetase activity are increased by salt-induced proteolysis in cashew leaves. Journal of Plant Physiology, 2003, 160(2): 115-123.
[12]Agata G, Domenica N, Angelica G, Antonio B. The glutamine synthetase (GS2) genes in relation to grain protein content of durum wheat. Functional and Integrative Genomics, 2011, 11: 665-670.
[13]Fuentes S I, Allen D J, Ortiz-Lopez A, Hernandez G. Over-expression of cytosolic glutamine synthetase inereases photosynthesis and growth at low nitrogen concentrations. Journal of Experimental Botany, 2001, 52: 1071-1081.
[14]Kozaki A, Takeba G. Photorespiration protein C3 plants from photooxidation. Nature, 1996, 384: 557-560.
[15]Oliveira I C, Brears T, Knight T J, Clark A, Coruzzi G M. Overexpression of cytosolic glutamine synthetase relation to nitrogen, light, and photorespiration. Plant Physiology, 2002, 129(3): 1170-1180.
[16]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 Molecular Biology, 2000, 43: 103-111.
[17]Cai H M, Zhou Y, Xiao J H, Li X H, Zhang Q F, Lian X M. Overexpression glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice. Plant Cell Reports, 2009, 28: 527-537.
[18]Liu T M, Zhu S Y, Tang Q M, Chen P, Yu Y T, Tang S W. De novo assemble and characterization of transcriptome using Illumina paired-end sequencing and identification of CesA gene in ramie (Boehmeria nivea L. Gaud). BMC Genomics, 2013, 14: 125-136.
[19]McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley  R. Leaf disc transformation of cultivated tomato (L. esculentum)  using Agrobacterium tumefaciens. Plant Cell Reports, 1986, 5(2): 81-84.
[20]Volkov R A, Panchuk I I, Schoffi F. Heat-stress-dependency and developmental modulation of gene expression: The potential of house-keeping genes as internal standards in mRNA expression profiling using real-time RT-PCR. Journal of Experimental Botany, 2003, 54: 2343-2349.
[21]Wang X, Replogle A, Davis E L, Mitchum M G. The tobacco Cel7 gene promoter is auxin-responsive and locally induced in nematode feeding sites of heterologous plants. Molecular Plant Pathology, 2007, 8: 423-436.
[22]Cai H, Zhou Y, Xiao J, Li X, Zhang Q, Lian X. Overexpression glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice. Plant Cell Reports, 2009: 527-537.
[23]Melo P M, Lima L M, Santos I M, Carvalho H G, Cullimore J V. Expression of the plastid-located glutamine synthetase of Medicago truncatula: Accumulation of the precursor in root nodules reveals an in vivo control at the level of protein import into plastids. Plant Physiology, 2003, 132: 390-399.
[24]Kanazin V, Talbert H, See D, DeCamp P, Nevo E, Blake T. Discovery and assay of single-nucleotide polymorphisms in barley (Hordeum vulgare). Plant Molecular Biology, 2002, 48(5): 529-537.
[25]Zhu Y L, Song Q J, Hyten D L, Van-Tossell C P, Matukumalli L K, Grimm D R, Hyatt S M, Fickus E W, Yong N D, Cregan P B. Single-nucleotide polymorphisms in soybean. Genetics, 2003, 163(3): 1123-1134.
[26]Morales M, Roig E, Monforte A J, Arus P, Garcia-Mas J. Single-nucleotide polymorphisms detected in expressed sequence tags of melon (Cucumis melo L.). Genome, 2004, 47(2): 352-360.
[27]Balley J, Barker G, O’Sullivan H, Edwards K J, Edwards D.   Mining for single nucleotide polymorphisms and insertion/ deletion in maize expressed sequence tag data. Plant Physiology, 2003, 132(1): 84-91.
[28]Nasu S, Suzuki J, Ohta R, Hasegawa K, Yui R, Kitazawa N, Monna L, Minobe Y. Search for and analysis of single nucleotide polymorphisms (SNPs) in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers. DNA Research, 2002, 9(5): 163-171.
[29]Bhattramakki D, Dolan M, Hanafey M, Wineland R, Vaske D, Register J C, Tingey S V, Rafalski A. Insertion-deletion polymorphisms in 3’-regions of maize genes occur frequently and can be used as highly informative genetic markers. Plant Molecular Biology, 2002, 48(5/6): 539-547.
[30]Hayashi K, Hashimoto N, Daigen M, Ashikawa I. Development  of PCR-based SNP markers for rice blast resistance genes at   the pizlocus. Theoretical and Applied Genetics, 2004, 108(7): 1212-1220.
[31]Umemoto T, Aoki N. single-nucleotide polymorphisms in rice starch synthase IIa that alter starch gelatinization and starch association of the enzyme. Functional Plant Biology, 2005, 32(9): 763-768.
[32]Spielmeyer M, Ellis M H, Chandler P M. Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellins 20-oxidase gene. Proceedings of the National Academy of Sciences of the USA, 2002, 99(13): 9043-9048.
[33]Hirel B, Gouis L J, Ney B, Gallais A. The challenge of improving nitrogen use efficiency in crop plants: Towards a more central role for genetic variability and quantitative genetics within integrated approaches. Journal of Experimental Botany, 2007, 58: 2369-2387.
[34]姜涛. 苎麻饲用资源产量与品质性状的研究[D]. 北京: 中国农业科学院, 2008.
Jiang T. Studies on yield and quality character of ramie[D]. Beijing: Chinese Academy of Agricultural Science, 2008. (in Chinese)
[35]Kumada Y, Benson D R, Hillemann D, Hosted T J, Rochefort D A, Thompson C J, Wohlleben W, Tateno Y. Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proceedings of the National Academy of Sciences of the USA, 1993, 90(7): 3009-3013.
[36]郭婷, 佘玮, 肖呈祥, 曹诣, 赵丹博, 崔国贤. 饲用苎麻研究进展. 作物研究, 2012, 26(6): 730-733.
Guo T, She W, Xiao C X, Cao Y, Zhao D B, Cui G X. Research progress of ramie for forage. Crop Research, 2012, 26(6): 730-733. (in Chinese)
[37]Downs C G, Borst W M, Hurst P L, Stevenson D G. Isoforms of glutamine synthetase in asparagus spears: The cytososic enzyme increases after harvest. Plant Cell Environment, 1994, 17: 1045-1052.
[38]Temple S J, Knight T J, Unkefer P J, Sengupta-Gopalan C. Modulation of glutamine synthetase gene expression in tobacco by the introduction of an alfalfa glutamine synthetase gene in sense and antisense orientation: Molecular and biochemical analysis. Molecular and General Genetics, 1993, 236: 315-325.
[39]Vincent R, Fraiser V, Chaillou S, Limani M A, Deleens E, Phillipson B, Douat C, Bontin J, Hirel B. Over expression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development. Planta, 1997, 201: 424-433.
[40]Fei H, Chaillou S, Hirel B, Mahon J D, Vessey K J. Overexpression of a soybean cytosolic glutamine synthetae gene linked to organ-specific promoters in pea plants grown in different concentration of nitrate. Planta, 2003, 216: 467-474.
[41]Ortega J L, Temple S J, Sengupta-Gopalan C. Constitutive over-expression of cytosolic glutamine synthetase (GS1) gene in transgenic alfalfa demonstrate that GS1 may be regulated at the level of RNA stability and protein turnover. Plant Physiology, 2001, 126: 109-121.
[42]Gallardo F, Fu J, Canton F R, Garcia-Gutierez A, Canovas F M, Kirby E G. Expression of a conifer glutamine synthetase gene in transgenic poplar. Planta, 1999, 210: 19-26.
[43]Man H, Boriel R, El-Khatib R, Kirby E G. Characterization of transgenic poplar with ectopic expression of pine cytosolic glutamine synthetase under conditions of varying nitrogen availability. New Phytologist, 2005, 167: 31-39.
[44]Migge A, Carrayol E, Hirel B, Becker T W. Leaf-specific overexpression of plastidic glutamine synthetase stimulates the growth of transgenic tobacco seedlings. Planta, 2000, 210: 252-260.
[45]Fu J, Sampalo R, Gallardo F, Canovas F M, Kirby E G. Assembly of a cytosolic pine glutamine synthetase holoenzyme in leaves of transgenic poplar leads to enhanced vegetative growth in young plants. Plant Cell Environment, 2003, 26: 411-418.
[1] ZHANG JianJun, DANG Yi, ZHAO Gang, WANG Lei, FAN TingLu, LI ShangZhong. Influences of Mulching Periods and Nitrogen Application Rates on Maize Yield as well as Water and Nitrogen Use Efficiencies in Loess Plateau of Eastern Gansu Province [J]. Scientia Agricultura Sinica, 2022, 55(3): 479-490.
[2] RU Chen,HU XiaoTao,LÜ MengWei,CHEN DianYu,WANG WenE,SONG TianYuan. Effects of Nitrogen on Nitrogen Accumulation and Distribution, Nitrogen Metabolizing Enzymes, Protein Content, and Water and Nitrogen Use Efficiency in Winter Wheat Under Heat and Drought Stress After Anthesis [J]. Scientia Agricultura Sinica, 2022, 55(17): 3303-3320.
[3] MA XueMeng,YU ChengMin,SAI XiaoLing,LIU Zhen,SANG HaiYang,CUI BaiMing. PSORA: A Strategy Based on High-Throughput Sequence for Analysis of T-DNA Insertion Sites [J]. Scientia Agricultura Sinica, 2022, 55(15): 2875-2882.
[4] LU BingLin,CHE ZongXian,ZHANG JiuDong,BAO XingGuo,WU KeSheng,YANG RuiJu. Effects of Long-Term Intercropping of Maize with Hairy Vetch Root Returning to Field on Crop Yield and Nitrogen Use Efficiency Under Nitrogen Fertilizer Reduction [J]. Scientia Agricultura Sinica, 2022, 55(12): 2384-2397.
[5] HE KeWei,CHEN JiaFa,ZHOU ZiJian,WU JianYu. Fusarium verticillioides Resistant Maize Inbred Line Development Using Host-Induced Gene Silencing Technology [J]. Scientia Agricultura Sinica, 2021, 54(9): 1835-1845.
[6] PENG BiLin,LI MeiJuan,HU XiangYu,ZHONG XuHua,TANG XiangRu,LIU YanZhuo,LIANG KaiMing,PAN JunFeng,HUANG NongRong,FU YouQiang,HU Rui. Effects of Simplified Nitrogen Managements on Grain Yield and Nitrogen Use Efficiency of Double-Cropping Rice in South China [J]. Scientia Agricultura Sinica, 2021, 54(7): 1424-1438.
[7] XIAO Fang,LI Jun,WANG HaoQian,ZHAI ShanShan,CHEN ZiYan,GAO HongFei,LI YunJing,WU Gang,ZHANG XiuJie,WU YuHua. Establishment and Application of A Duplex ddPCR Method to Quantify the NK603/zSSIIb Copy Number Ratio in Transgenic Maize NK603 [J]. Scientia Agricultura Sinica, 2021, 54(22): 4728-4739.
[8] JIN Rong,LIU Ming,ZHAO Peng,ZHANG QiangQiang,ZHANG AiJun,TANG ZhongHou. IbMKP6, A Mitogen-Activated Protein Kinase, Confers Low Temperature Tolerance in Sweetpotato [J]. Scientia Agricultura Sinica, 2021, 54(20): 4265-4273.
[9] SONG ShaoZheng,YU KangYing,ZHANG Ting,LU Rui,PAN ShengQiang,CHENG Yong,ZHOU MingMing. Preparation and Expression of rhPA/GH Double Transgenic Rabbits [J]. Scientia Agricultura Sinica, 2021, 54(2): 412-421.
[10] QU XiaoLing,JIAO YuBing,LUO JianDa,SONG LiYun,LI Ying,SHEN LilLi,YANG JinGuang,WANG FengLong. Cloning of Nicotiana benthamiana NAC062 and Its Inhibitory Effect on Potato Virus Y Infection [J]. Scientia Agricultura Sinica, 2021, 54(19): 4110-4120.
[11] LONG Qin,DU MeiXia,LONG JunHong,HE YongRui,ZOU XiuPing,CHEN ShanChun. Effect of Transcription Factor CsWRKY61 on Citrus Bacterial Canker Resistance [J]. Scientia Agricultura Sinica, 2020, 53(8): 1556-1571.
[12] HuiLin YU,Fang JIA,ZongHua QUAN,HaiLan CUI,XiangJu LI. Effects of Glyphosate on Weed Control, Soybean Safety and Weed Occurrence in Transgenic Herbicide-Resistant Soybean [J]. Scientia Agricultura Sinica, 2020, 53(6): 1166-1177.
[13] XIANG Wei,WANG Lei,LIU TianQi,LI ShiHao,ZHAI ZhongBing,LI ChengFang. Effects of Biochar Plus Inorganic Nitrogen on the Greenhouse Gas and Nitrogen Use Efficiency from Rice Fields [J]. Scientia Agricultura Sinica, 2020, 53(22): 4634-4645.
[14] DING XiangPeng,LI GuangHao,ZHANG JiWang,LIU Peng,REN BaiZhao,ZHAO Bin. Effects of Base Application Depths of Controlled Release Urea on Yield and Nitrogen Utilization of Summer Maize [J]. Scientia Agricultura Sinica, 2020, 53(21): 4342-4354.
[15] ZOU WenXiu,HAN XiaoZeng,LU XinChun,CHEN Xu,HAO XiangXiang,YAN Jun. Effect of Maize Straw Return Aftereffect on Nitrogen Use Efficiency of Maize [J]. Scientia Agricultura Sinica, 2020, 53(20): 4237-4247.
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