|
|
|
|
|
| Molecular Cloning and Functional Characterization of a Salt Tolerance- Associated Gene IbNFU1 from Sweetpotato |
| WANG Lian-jun, HE Shao-zhen, ZHAI Hong, LIU De-gao, WANGYan-nan , LIU Qing-chang |
| Key Laboratory of Crop Genomics and Genetic Improvement, Ministry of Agriculture/Beijing Key Laboratory of Crop Genetic Improvement/ Laboratory of Crop Heterosis and Utilization, Ministry of Education/China Agricultural University, Beijing 100193, P.R.China |
|
|
|
摘要 Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. Based on the EST sequence selected from salt-stressed suppression subtractive hybridization (SSH) cDNA library constructed with a salt-tolerant mutant LM79, a NFU gene, termed IbNFU1, was cloned from sweetpotato (Ipomoea batatas (L.) Lam.) via rapid amplification of cDNA ends (RACE). The cDNA sequence of 1 117 bp contained an 846 bp open reading frame encoding a 281 amino acids polypeptide with a molecular weight of 30.5 kDa and an isoelectric point (pI) of 5.12. IbNFU1 gene contained a conserved Cys-X-X-Cys motif in C-terminal of the iron-sulfur cluster domain. The deduced amino acid sequence had 66.08 to 71.99% sequence identity to NFU genes reported in Arabidopsis thaliana, Eucalyptus grandis and Vitis vinifera. Real-time quantitative PCR analysis revealed that the expression level of IbNFU1 gene was significantly higher in the roots of the mutant LM79 compared to the wild-type Lizixiang. Transgenic tobacco (cv. Wisconsin 38) plants expressing IbNFU1 gene exhibited significantly higher salt tolerance compared to the untransformed control plants. It is proposed that IbNFU1 gene has an important function for salt tolerance of plants.
Abstract Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. Based on the EST sequence selected from salt-stressed suppression subtractive hybridization (SSH) cDNA library constructed with a salt-tolerant mutant LM79, a NFU gene, termed IbNFU1, was cloned from sweetpotato (Ipomoea batatas (L.) Lam.) via rapid amplification of cDNA ends (RACE). The cDNA sequence of 1 117 bp contained an 846 bp open reading frame encoding a 281 amino acids polypeptide with a molecular weight of 30.5 kDa and an isoelectric point (pI) of 5.12. IbNFU1 gene contained a conserved Cys-X-X-Cys motif in C-terminal of the iron-sulfur cluster domain. The deduced amino acid sequence had 66.08 to 71.99% sequence identity to NFU genes reported in Arabidopsis thaliana, Eucalyptus grandis and Vitis vinifera. Real-time quantitative PCR analysis revealed that the expression level of IbNFU1 gene was significantly higher in the roots of the mutant LM79 compared to the wild-type Lizixiang. Transgenic tobacco (cv. Wisconsin 38) plants expressing IbNFU1 gene exhibited significantly higher salt tolerance compared to the untransformed control plants. It is proposed that IbNFU1 gene has an important function for salt tolerance of plants.
|
|
Received: 11 August 2011
Accepted:
|
| Fund: This work was supported by the China Agricultural Research System (Sweetpotato), the National High-Tech Res e a rch and Deve lopment Program of Chin a (2009AA10Z102), the National Transgenic Plants Project of China (2009ZX08009-064B), and the National Natural Science Foundation of China (31071468). |
|
Corresponding Authors:
Correspondence LIU Qing-chang, Tel/Fax: +86-10-62733710, E-mail: liuqc@cau.edu.cn
E-mail: liuqc@cau.edu.cn
|
Cite this article:
WANG Lian-jun, HE Shao-zhen, ZHAI Hong, LIU De-gao, WANGYan-nan , LIU Qing-chang.
2013.
Molecular Cloning and Functional Characterization of a Salt Tolerance- Associated Gene IbNFU1 from Sweetpotato. Journal of Integrative Agriculture, 12(1): 27-35.
|
| [1]Bae J M, Liu J R. 1997. Molecular cloning andcharacterization of two novel isoforms of the smallsubunit of ADP glucose pyrophosphorylase from sweetpotato. Molecular and General Genetics, 254, 179-185[2]Balasubramanian R, Shen G Z, Bryant D A, Golbeck J H.2006. Regulatory roles for IscA and SufA in ironhomeostasis and redox stress responses in thecyanobacterium Synechococcus sp. strain PCC 7002.Journal of Bacteriology, 188, 3182-3191[3]Chen C J, Huang C Y, Huang J K, Lin C Y, Lin C T. 2009.Cloning, expression, and purification of a functionalglutathione reductase from sweet potato (Ipomoeabatatas L. Lam): kinetic studies and characterization.Journal of Agricultural and Food Chemistry, 57, 4403-4408[4]Chen H J, Hou WC, Yang C Y, Huang D J, Liu J S, Lin Y H.2003. Molecular cloning of two metallothionein-likeprotein genes with differential expression patterns fromsweet potato (Ipomoea batatas) leaves. Journal ofPlant Physiology, 160, 547-555[5]Chen H J, Huang D J, Hou W C, Liu J S, Lin Y H. 2006.Molecular cloning and characterization of a granulincontainingcysteine protease SPCP3 from sweet potato(Ipomoea batatas) senescent leaves. Journal of PlantPhysiology, 163, 863-876[6]Chen H J, Su C T, Lin C H, Huang G J, Lin Y H. 2010.Expression of sweet potato cysteine protease SPCP2 altered developmental characteristics and stressresponses in transgenic Arabidopsis plants. Journalof Plant Physiology, 167, 838-847[7]Chen G H, Huang L T, Yap MN, Lee R H, Huang Y J, ChengM C, Chen S C G. 2002. Molecular characterization of asenescence-associated gene encoding cysteineproteinase and its gene expression during leafsenescence in sweet potato. Plant Cell Physiology,43, 984-991[8]Duncan D B. 1955. Multiple range and multiple F tests.Biometrics, 11, 1-42[9]Durmus A, Eicken C, Spener F, Krebs B. 1999. Cloning andcomparative protein modeling of two purple acidphosphatase isozymes from sweet potatoes (Ipomoeabatatas). Biochimica et Biophysica Acta, 1434, 202-209[10]Fernández-Falcón M, Hernández M, Alvarez C E, Borges AA. 2006. Variation in nutrition along time and relativechlorophyll content of Leucospermum cordifolium cv.‘High Gold’, and their relationship with chloroticsypmptoms. Scientia Horticulturae, 107, 373-379[11]Gorantla M, Babu P R, Lachagari V B R, Feltus FA, PatersonA H, Reddy A R. 2005. Functional genomics of droughtstress response in rice: tanscript mapping of annotatedunigenes of an indica rice (Oryza sativa L. cv. Nagina22). Scientia Horticulturae, 89, 496-514[12]Hamada T, Kim S H, Shimada T. 2006. Starch-branchingenzyme I gene (IbSBEI) from sweet potato (Ipomoeabatatas): molecular cloning and expression analysis.Biotechnology Letters, 28, 1255-1261[13]He S Z, Han Y F, Wang Y P, Zhai H, Liu Q C. 2009. In vitroselection and identification of sweetpotato (Ipomoeabatatas (L.) Lam.) plants tolerant to NaCl. Plant CellTissue and Organ Culture, 96, 69-74[14]Horsch R B, Fry J E, Hoffmann N L, Eichholtz D, Rogers SG, Fraley R T. 1985. A simple and general method fortransferring genes into plants. Science, 227, 1229-1231[15]Huang D J, Chen H J, Hou WC, Chen T E, Hsu W Y, Lin YH. 2005a. Expression and function of a cysteineproteinase cDNA from sweet potato (Ipomoea batatas[L.] Lam. ‘Tainong 57’) storage roots. Plant Science,169, 423-431[16]Huang D J, Chen H J, Hou WC, Lin Y H. 2004. Isolation andcharacterization of thioredoxin h cDNA from sweetpotato (Ipomoea batatas [L.] Lam. ‘Tainong 57’) storageroots. Plant Science, 166, 515-523[17]Huang D J, Chen H J, Lin Y H. 2005b. Isolation andexpression of protein disulfide isomerase cDNA fromsweet potato (Ipomoea batatas [L.] Lam. ‘Tainong 57’)storage roots. Plant Science, 169, 776-784[18]Jefferson, R A, Kavanagh T A, Bevan M W. 1987. GUSfusions: ?-glucuronidase as a sensitive and versatilegene fusion marker in higher plants. The EMBOJournal, 6, 3901-3907[19]Kaakoush N O, Raftery M, Mendz G L. 2008. Molecularresponses of Campylobacter jejuni to cadmium stress.FEBS Journal, 275, 5021-5033[20]Kim K Y, Huh G H, Lee H S, Kwon S Y, Hur Y, Kwak S S.1999. Molecular characterization of cDNAs for twoanionic peroxidases from suspension cultures of sweetpotato. Molecular and General Genetics, 261, 941-947[21]Kumar K R R, Kirti P B. 2011. Differential gene expressionin Arachis diogoi upon interaction with peanut lateleaf spot pathogen, Phaeoisariopsis personata andcharacterization of a pathogen induced cyclophilin.Plant Molecular Biology, 75, 497-513[22]Kumeta H, Ogura K, Asayama M, Katoh S, Katoh E,Teshima K, Inagaki F. 2007. The NMR structure of thedomain II of a chloroplastic NifU-like protein OsNifU1A.Journal of Biomolecular NMR, 38, 161-164[23]Lalusin A G, Nishita K, Kim S, Ohta M, Fujimura T. 2006. Anew MADS-box gene (IbMADS10) from sweet potato(Ipomoea batatas (L.) Lam) is involved in theaccumulation of anthocyanin. Molecular Genetics andGenomics, 275, 44-54[24]Léon S, Touraine B, Ribot C, Briat J, Lobréaux S. 2003. Ironsulphurcluster assembly in plants: distinct NFU proteinsin mitochondria and plastids from Arabidopsisthaliana. Biochemical Journal, 371, 823-830[25]Lin C T, Yeh K W, Kao M C, Shaw J F. 1993. Cloning andcharacterization of a cDNA encoding the cytosoliccopper/zinc-superoxide dismutase from sweet potatotuberous root. Plant Molecular Biology, 23, 911-913[26]Lorenzo L D, Merchan F, Laporte P, Thompson R, Clarke J,Sousa C, Crespi M. 2009. A novel plant leucine-richrepeat receptor kinase regulates the response ofMedicago truncatula roots to salt stress. The PlantCell, 212, 668-680[27]Mohammadi M, Kav N N V, Deyholos M K. 2008.Transcript expression profile of water-limited roots ofhexaploid wheat (Triticum aestivum ‘Opata’). Genome,51, 357-367[28]Pereira P M, He Q, Xavier A V, Zhou J Z, Pereira I A C,Louro R O. 2008. Transcriptional response ofDesulfovibrio vulgaris Hildenborough to oxidativestress mimicking environmental conditions. Archivesof Microbiology, 189, 451-461[29]Schilke B, Voisine C, Beinert H, Craig E. 1999. Evidence fora conserved system for iron metabolism in themi tochondria of Saccharomyces cerevi siae.Proceedings of the National Academy of Sciences ofthe United States of America, 96, 10206-10211[30]Schmittgen T D, Livak K J. 2008. Analyzing real-time PCRdate by the comparative CT method. Nature Protocols,3, 1101-1108[31]Seo S G, Kim J S, Kang S W, Shin M R, Yang Y S, Lee G P,Hong J S, Kim S H. 2008. Touch-induced gene (IbTCH1)from sweet potato [Ipomoea batatas (L.) Lam.]:molecular cloning and functional analysis. PlantBiotechnology Reports, 2, 253-258[32]Shim K S, Cho S K, Jeung J U, Jung K W, You M K, Ok S H,Chung Y S, Kang H K, Hwang H G, Choi H C, et al. 2004. Identification of fungal (Magnaporthe grisea) stressinducedgenes in wild rice (Oryza minuta). Plant CellReport, 22, 599-607[33]Tanaka M, Yasuhiro T, Nakayama H, Nakatani M, TaharaM. 2009. Altered carbohydrate metabolism in thestorage roots of sweetpotato plants overexpressing theSRF1 gene, which encodes a Dof zinc fingertranscription factor. Planta, 230, 737-746[34]TongW H, Jameson G N L, Huynh B H, Rouault T A. 2003.Subcellular compartmentalization of human Nfu, an ironsulfurcluster scaffold protein, and its ability toassemble a [4Fe-4S] cluster[35]Proceedings of theNational Academy of Sciences of the United States ofAmerica, 100, 9762-9767[36]Touraine B, Boutin J P, Marion-Poll A, Briat J F, Peltier G,Lobréaux S. 2004. Nfu2, a scaffold protein required for[4Fe-4S] and ferredoxin iron-sulphur cluster assemblyin Arabidopsis chloroplasts[37]The Plant Journal, 40,101-111[38]Wang H Y, Huang Y C, Chen S F, Yeh KW. 2003. Molecularcloning, characterization and gene expression of a waterdeficiency and chilling induced proteinase inhibitor Igene family from sweet potato (Ipomoea batatas Lam.)leaves. Plant Science, 165, 191-203[39]Wang S J, Yeh K W, Tsai C Y. 1999. Molecularcharacterization and expression of starch granuleboundstarch synthase in the sink and source tissuesof sweet potato. Physiologia Plantarum, 106, 253-261[40]Wang W X, Vinocur B, Altman A. 2003. Plant responses todrought, salinity and extreme temperatures, towardsgenetic engineering for stress tolerance. Planta, 218,1-14[41]Xu K, He B, Zhou S, Li Y, Zhang Y Z. 2010. Cloning andcharacterization of the Rubisco activase gene fromIpomoea batatas (L.) Lam. Molecular Biology Reports,37, 661-668[42]Yabe T, Morimoto K, Kikuchi S, Nishio K, Terashima I,Nakai M. 2004. The Arabidopsis chloroplastic NifUlikeprotein CnfU, which can act as an iron-sulfur clusterscaffold protein, is required for biogenesis of ferredoxinand photosystem I. The Plant Cell, 16, 993-1007[43]Yabe T, Yamashita E, Kikuchi A, Morimoto K, Nakagawa A,Tsukihara T, Nakai M. 2008. Structural analysis ofArabidopsis CnfU protein, an iron-sulfur clusterbiosynthetic scaffold in chloroplasts. Journal ofMolecular Biology, 381, 160-173[44]Yale J, Bohnert H J. 2001. Transcript expression inSaccharomyces cerevisiae at high salinity. The Journalof Biological Chemistry, 276, 15996-16007[45]Yap M N, Lee R H, Huang Y J, Liao C J, Chen S C G. 2003.Molecular characterization of a novel senescenceassociatedgene SPA15 induced during leaf senescencein sweet potato. Plant Molecular Biology, 51, 471-481[46]Zhou W, Huang C T, Gong Y F, Feng Q L, Gao F. 2010.Molecular cloning and expression analysis of an ANSgene encoding anthocyanidin synthase from purplefleshedsweet potato [Ipomoea batatas (L.) Lam]. PlantMolecular Biology Reporter, 28, 112-121 |
| No Suggested Reading articles found! |
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
