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leaa 氨基酸-概述说明以及解释1.引言1.1 概述概述:氨基酸是构成蛋白质的基本单元，是生命体内重要的有机分子之一。
它们在生物体内承担着多种生物化学功能，包括构建细胞结构、催化生物化学反应、传递信号等。
氨基酸的种类繁多，每一种氨基酸都具有特定的结构和性质，不同的氨基酸组合形成了不同的蛋白质，进而决定了生物体的生理功能。
本文旨在系统介绍氨基酸的定义、分类和生物功能，探讨氨基酸在生物体内的重要性，并展望氨基酸在未来研究和应用中的发展前景。
通过深入了解氨基酸这一重要的生物分子，我们可以更好地理解生命的奥秘，促进生物化学领域的研究和应用。
1.2 文章结构文章结构部分的内容:本文主要分为引言、正文和结论三个部分。
在引言部分，我们将简要介绍氨基酸的概念、文章结构和研究目的。
在正文部分，我们将详细探讨氨基酸的定义、分类以及在生物体中的重要功能。
最后在结论部分，我们将对氨基酸的研究进行总结，并展望未来氨基酸在生物体中的重要性。
希望通过本文的阐述，读者能够更加全面地了解和认识氨基酸这一重要的生物分子。
1.3 目的本文旨在深入探讨氨基酸这一生物学中至关重要的分子，包括其定义、分类和生物功能等方面。
通过对氨基酸的全面了解，我们可以更好地理解其在生物体内的作用和重要性，为进一步研究和应用氨基酸提供基础和指导。
同时，我们也希望通过本文的撰写，使读者对氨基酸有一个全面而清晰的认识，增强对生物学领域的理解和认识。
在文章的结论部分，我们将总结氨基酸的重要性和生物功能，并展望未来在氨基酸研究领域的发展前景。
通过本文的阅读，读者将对氨基酸有一个更深入的了解，并对生命的奥秘有更多的探索和思考。
2.正文2.1 氨基酸的定义氨基酸是构成蛋白质的基本单位之一，是生命体内必不可少的有机分子。
它们是由氨基基团（NH2）和羧基团（COOH）组成的。
氨基酸还含有一个特定的侧链，不同氨基酸的侧链结构不同，决定了氨基酸的性质和功能。
氨基酸通过肽键连接形成蛋白质。
ORIGINAL PAPERMolecular characterization and functional analysisby heterologous expression in E.coli under diverse abiotic stresses for OsLEA5,the atypical hydrophobic LEA protein from Oryza sativa L.Shuai He •Lili Tan •Zongli Hu •Guoping Chen •Guixue Wang •Tingzhang HuReceived:4June 2011/Accepted:12November 2011/Published online:30November 2011ÓSpringer-Verlag 2011Abstract In this study,we report the molecular charac-terization and functional analysis of OsLEA5gene,which belongs to the atypical late embryogenesis abundant (LEA)group 5C from Oryza sativa L.The cDNA of OsLEA5contains a 456bp ORF encoding a polypeptide of 151amino acids with a calculated molecular mass of 16.5kDa and a theoretical pI of 5.07.The OsLEA5polypeptide is rich in Leu (10%),Ser (8.6%),and Asp (8.6%),while Cys,Trp,and Gln residue contents are very low,which are 2, 1.3,and 1.3%,respectively.Bioinformatic analysis revealed that group 5C LEA protein subfamily contains a Pfam:LEA_2domain architecture and is highly hydro-phobic,intrinsically ordered with largely b -sheet and spe-cific amino acid composition and distribution.Real-time PCR analysis showed that OsLEA5was expressed in dif-ferent tissue organs during different development stages of rice.The expression levels of OsLEA5in the roots and panicles of full ripe stage were dramatically increased.The results of stress tolerance and cell viability assay demon-strated that recombinant E.coli cells producing OsLEA5fusion protein exhibited improved resistance against diverse abiotic stresses:high salinity,osmotic,freezing,heat,and UV radiation.The OsLEA5protein confers sta-bilization of the LDH under different abiotic stresses,such as heating,freeze–thawing,and drying in vitro.The com-bined results indicated that OsLEA5protein was a hydro-phobic atypical LEA and closely associated with resistance to multiple abiotic stresses.This research offered the valuable information for the development of crops with enhanced resistance to diverse stresses.Keywords Abiotic stresses ÁHeterologous expression ÁHydrophobic ÁLate embryogenesis abundant proteins ÁOryza sativa L.IntroductionFor a long time,higher plants have developed multi-path-way,multilevel,and multi-scale survival strategies for continual changes in response to adverse conditions (Scoltis and Soltis 2003).The evolution of late embryo-genesis abundant (LEA)proteins is one of these changes.LEA protein family is a large protein family that is closely associated with resistance to abiotic stresses in organisms (Boucher et al.2010;Su et al.2011;Tunnacliffe and Wise 2007).LEA protein was originally identified three decades ago in orthodox seeds during the maturation phase of embryogenesis (Dure et al.1981).Until now,great quantity novel LEA proteins have been characterized in maturing seeds and anhydrobiotic plants,animals,and microorgan-isms (Wise and Tunnacliffe 2004).Usually,accumulation of LEA proteins during mid to late embryogenesis corre-lates with increased levels of the phytohormone abscisic acid (ABA)and with the acquisition of dehydration toler-ance,which are also induced by freezing,high tempera-ture,high salinity,and osmotic stress (Su et al.2011;Communicated by R.Hagemann.Nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under the accession number JF776156.Electronic supplementary material The online version of this article (doi:10.1007/s00438-011-0660-x )contains supplementary material,which is available to authorized users.S.He ÁL.Tan ÁZ.Hu ÁG.Chen ÁG.Wang ÁT.Hu (&)Key Laboratory of Biorheological Science and Technology,Ministry of Education,College of Bioengineering,Chongqing University,Chongqing 400044,China e-mail:tzhu2002@Mol Genet Genomics (2012)287:39–54DOI 10.1007/s00438-011-0660-xTunnacliffe and Wise2007).Upon germination these proteins are rapidly degraded(Bartels et al.1988).LEA proteins are mainly localized in cytoplasm,mitochondrion, or nuclear regions(Zhang et al.2002).They are relatively small proteins with low molecular weight ranging mainly from10to30kDa(He and Fu1996).Most common classifications are related to protein chemical characteristics.The traditional criterion was on the basis of amino acid sequence homology from different plant species and conserved motifs which presumably undertake different functions during the periods of water deficit.At present,the terminology of LEA proteins varies between different articles.Table1compares classification systems proposed by Dure,Bray,Bies-Ethe`ve,and Batta-glia,respectively(Battaglia et al.2008;Bies-Ethe`ve et al. 2008;Bray1993;Dure et al.1989).In this work,we adopt the nomenclature introduced by Battaglia,and LEA proteins are categorized into seven distinctive groups (Battaglia et al.2008).Groups1,2,3,4,6,and7,which share specific motifs within each respective group,are considered as typical or genuine LEA protein.While group 5lack significant signature motifs or consensus sequences, it is considered as an atypical LEA protein.Group5LEA proteins contain a significantly higher proportion of hydrophobic residues than typical LEA proteins and are hydrophobic proteins(Battaglia et al.2008).Compared with the categorical groups mentioned above,group5is relatively vague and lax.Therefore,this group has non-homologous proteins.The group5can further be divided into subgroups5A(SMP),5B(LEA_3),and5C(LEA_2) according to their Pfam(Table1).Cotton gD34,soybean GmPM25,Arabidopsis PAP140,ATDI21,SAG21,Prosopis juliflora LEA3,Picea glauca EMB11,tomato ER5,and Lemmi9are involved in group5proteins.As the most distinctive set of LEA proteins,in view of their physico-chemical properties,they show significant differences compared with the canonical LEA protein.Group5LEA proteins display relatively high hydrophobicity(Maitra and Cushman1994),which is precisely the opposite with the extensive hydrophilic characteristics of other LEA pro-teins.An additional peculiarity of the group5LEA pro-teins is that they are not soluble after boiling,suggesting that they are apparently not heat stable.Noteworthy,group 5LEA proteins are natively folded(Hundertmark and Hincha2008).Fourier-transformed infrared(FTIR)spec-troscopy shows that they contain a large quantity of b-sheet rather than a-helixed structure in the hydrated state(Boudet et al.2006).Currently,the group5C protein LEA14-A from Arabidopsis is the only LEA protein which was proven to have a defined secondary and tertiary structure in solution of the LEA proteins(Singh et al.2005).All of these dif-ferences imply that they are probably functionally different from the typical proteins.Therefore,a question was raised: whether this intriguing group of proteins should in the end be regarded as LEA proteins(Boucher et al.2010; Tunnacliffe and Wise2007).Although apparent similarities have not been detected between the members of the different LEA protein fami-lies,in general,hydrophilicity,cytosolic,thermal stability, high content of Gly,Ala,Ser,large net charge and intrin-sically unstructured in solution,are their unifying and outstanding characteristics(Battaglia et al.2008;Wise and Tunnacliffe2004).Advanced structure of such protein contains nonperiodic linear and a-helixed structure without thermal dominative state and the corresponding dehydrated proteins exist in a natural form of dimers(Hong-Bo et al. 2005).Despite the marked absence of a defined tertiary structure,a variety of roles of LEA proteins was proposed, such as they act as hydration buffer,molecular shield, antioxidant,metal ion binding and sequestering,and another important role might be their contribute to the formation of tight glass matrices together with sugars inTable1Different nomenclatures of the given LEA protein groupsPfam no.Pfam Dure Battaglia a Bray Bies-Ethe`ve Representative proteinPF00477LEA_5D-19Group1Group1Group1Wheat TaEmPF00257Dehydrin D-11Group2Group2Group2Tomato Dhn1,soybean PM12 PF02987LEA_4D-7or D-29Group3Group3or5Group3Carrot ECP63,barley HVA1 PF03760LEA_1D-113Group4Group4Group4Tomato le25PF04927SMP D-34Group5A Group6Group5Carrot ECP31PF03242LEA_3D-73Group5B–Group6Arabidopsis ATDI21PF03168LEA_2D-95Group5C–Group7Cotton LEA14-APF10714LEA_6–Group6–Group8Kidney bean PvLEA18PF02496ABA_WDS–Group7––Tomato Asr1,pine lp3-3a The nomenclature used in this work–Not been classified by authordesiccated cells(Buitink and Leprince2004).Thus,it has been speculated that LEA proteins mainly function in the protection of membrane integrity and macromolecular structures or ameliorate the effect of drought stress by re-naturing unfolded proteins and maintaining minimum cel-lular-water requirements.Increasing evidence has indicated a positive correlation between the LEA proteins and abiotic stress tolerance particularly dehydration(Dure et al.1989; Tunnacliffe and Wise2007).However,in most cases their precise functions have not yet been elucidated clearly and rely almost solely on deductions from expression data.In our ongoing research,we isolated,molecularly characterized and functionally analyzed the cDNA clone of a novel member of the atypical group5LEA protein from rice,namely OsLEA5.In order to make sense of the various defined functions of this enigmatic protein,the expression pattern of OsLEA5in rice was analyzed,the positive effect of OsLEA5on bacterial cell resistance to stresses was examined,and the protective effect of purified OsLEA5 protein on lactate dehydrogenase(LDH)activity was tested under different stresses in vitro.Materials and methodsPlant materials and treatmentsThe seeds of Oryza sativa cv.Zhonghua11were sterilized, soaked in water at28°C for2days and then hydroponically grown at28°C under16/8h light/dark photoperiod at an intensity of250l E/m2/s.The roots and leaves from rice plant at the seedling stage;their roots,stems,and leaves at tillering stage;and their roots,stems,leaves,and panicles at the heading stage,filling stage,and full ripe stage were collected and frozen in liquid nitrogen,then stored at -80°C,respectively.Isolation of OsLEA5cDNATotal RNA was extracted from the seedling using the Trizol reagent(Invitrogen,USA).One microgram of total RNA was used forfirst-strand cDNA synthesis,and the reverse transcription reactions were performed using oli-go(dT)18as30primer following the manufacturer’s proto-col.To get the complete ORF of OsLEA5cDNA,a pair of PCR primers was designed based on the predicted nucle-otide sequence of OsLEA5cDNA(NP_001056449).The sense primer was50-GATAATTTCACTCCAGCGTGC-30 and the antisense primer was50-CGGAGTTGGTTGATGA GATTA-30.The amplicon obtained by RT-PCR was cloned into pMD18-T vector(Takara,Dalian,China),then intro-duced into E.coli DH5a and sequenced(Invitrogen, Shanghai,China).In silico analysis of protein sequencesClustalW2.0and GeneDoc softwares were used for amino acid comparison and multiple alignments(http://www. /Tools/msa/clustalw2/).Sequence identities and similarities were determined using the BLAST program and the GenBank database on the NCBI web server.Motif analysis was performed using the Pfam program(http:// /Tools/InterProScan/).Secondary structure predictions were run with the DSSP,PSIPRED(http:// www.ibi.vu.nl/programs)(Jones1999;Kabsch and Sander 1983),PDISORDER(),and DisEMBL(http://dis.embl.de/)programs(Linding et al. 2003).The isoelectric point and molecular mass predictions were made using compute pI/Mw tool(/ tools/pi_tool.html).Analysis of protein hydropathy was done by constructing hydropathy plots with Kyte and Doolittle algorithm(http://ipsort.hgc.jp/)(Kyte and Doolittle1982). The grand average of hydropathy(GRAVY)and instability index of deduced proteins as well as the signal peptide and subcellular localization were predicted using the SoftBerry (),ProtParam(http://au.expasy. org/tools/protparam.html),and PSORT(http://psort.nibb. ac.jp)programs.Phylogenetic tree was performed with the neighbor-joining method using the MEGA4.0program (Saitu and Nei1987).Real-time PCR analysisTotal RNA from the samples was isolated and subsequently treated with DNase I.The RNAs were reverse transcribed using the Superscript TM III RNase H-Reverse Transcriptase kit(Invitrogen,USA).The synthesized cDNAs were diluted five times with water.qPCR DNA amplification and anal-ysis was carried out using the CFX96TM Real-Time System (C1000TM thermal cycler).All reactions were performed with OsLEA5qf(50-GATAATTTCACTCCAGCGTGC-30) and OsLEA5qr(50-TGTAGGTGAGCTCGCAGATG-30) using the SsoFast TM EvaGreenÒSupermix according to the manufacturer’s instructions.Reactions were performed in triplicate using5l l SsoFast EvaGreen Supermix,0.5l M each primer,1l l diluted cDNA,and RNase/DNase-free water to afinal volume of10l l.PCR amplification was performed using two-step cycling conditions of98°C for 3min,followed by40cycles of98°C for2s,and58°C for 10s.Amplification was followed by a melting curve anal-ysis with continualfluorescence data acquisition during the 65–95°C melt.Melt curve analysis of qPCR samples revealed that there was only one product for each gene primer reaction.The PCR products were sequenced to confirm the specific amplifications.The gene expression was normalized to OsEF1a,which was amplified withprimers OsEF1a f(50-ACATTGCCGTCAAGTTTGCTG-30) and OsEF1a r(50-AACAGCCACCGTTTGCCTC-30). Construction of the OsLEA5overexpression vectorIn order to construct the expression vector pET32a-OsLEA5, primers with restriction enzyme sites were designed: 50-CATGCCATGGGCATGTCGAGCTTGATGGAC-30 (Nco I site underlined)and50-CCCAAGCTTCGGAGTTGG TTGATGAGA-30(Hin dIII site underlined).The PCR products were cloned into pET32a vector at Nco I-Hin dIII site to construct the expression vector pET32a-OsLEA5, which express OsLEA5protein fused with TrxÁTag TM thio-redoxin at the N-terminus.The PCR-derived DNA clone was sequenced.pET32a-OsLEA5plasmid and the empty vector of pET32a were transformed into applicable E.coli host strain BL21,respectively.The recombinant pET32a-OsLEA5was obtained.The transformant of pET32a was used as a control.Expression,identification,and purification of OsLEA5 fusion protein in E.coliTransformed E.coli BL21cells carrying pET32a-OsLEA5 and pET-32a were grown in LB(Luria-Bertani)liquid medium supplemented with100l g/ml of ampicillin at 37°C about16h.The bacterial cultures were diluted 100-fold using fresh liquid LB,and allowed to incubate for 2–3h at37°C until exponential growth phase(OD600= 0.6–0.8).Isopropylthio-b-D-galactoside(IPTG)was then added into the cell cultures to afinal concentration of 0.8mM to induce expression of the inserted gene in recombinants,and further grown at37°C for4h.The E.coli cells were harvested by centrifugation and resus-pended in buffer(100mM NaCl,1mM EDTA,50mM pH 8.0Tris–HCl),and then lysed by ultrasonic fragmentation. The lysates were centrifuged again to collect supernatants. The soluble fraction were mixed with SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electropheresis)sample loading buffer(59)and boiled for5min.The overex-pressed proteins could be detected by SDS-PAGE according to standard methods.The OsLEA5protein was purified using HiTrap TM chelating HP column containing 1ml of Ni-IDA agarose resin(Amersham Biosciences)by following the manufacturer’s protocol.Protein concentra-tion in crude extracts was determined following the Brad-ford method(Bradford1976).Assay for abiotic stress tolerance of E.coli transformantsIPTG induction and cell cultures were prepared as descri-bed above.The concentration of all induced cultures in liquid LB was adjusted to OD600value of1.0.To measure responses to high salinity stress,the samples were diluted by tenfold with fresh LB medium supplemented with ampicillin at100l g/ml.One hundred microliters of the diluted sample were spotted on LB agar plates with 0.8mM IPTG,and containing additional100,200,300, 400,500,and600mM concentration gradient of KCl, 500mM NaCl,130mM MgCl2,and130mM CaCl2, respectively.To measure responses to hyperosmotic stress, 100l l of the diluted tenfold sample were plated on LB agar plates with0.8mM IPTG,containing additional600, 800,1,000,1,200,1,400,and1,600mM sorbitol,respec-tively.For the assay of the thermophylactic experiments, 1ml samples were transferred immediately to50°C.Ali-quots(100l l)were taken after0.5,1,1.5,2,2.5,and3h successively,diluted tenfold,and then100l l of dilutions were spread onto IPTG LB agar plates.For the assay of cryophylactic experiments,1ml samples in a microfuge tube were immediately placed in the-80°C freezer.Each day the samples were kept frozen for22.5h and allowed to thaw at ambient temperature(about30°C)for1.5h,which constituted one freeze–thaw cycle(Sleight et al.2006). Aliquots(100l l)were taken after2,4,6,8,10,and12 cycles successively,diluted tenfold,and then100l l of diluted samples were spread onto IPTG LB agar plates.To quantitate responses to ultraviolet(UV)radiation stress, 100l l diluted tenfold samples were plated on IPTG LB agar plates and directly exposed to ultraviolet radiation at a wave length of254nm(80l W/cm2)for5,7.5,10,12.5, 15,and20min separately.Following the above treatments, the stressed cells spread on IPTG solid medium were incubated for24–48h at37°C.Simultaneously,100l l of the corresponding untreated samples were plated on unstressed IPTG LB agar plates as a control.After incu-bation,the plates were scanned and cell viability was estimated by calculating the number of colony-forming units.Cell viability was plotted as the percentage of col-ony-forming units on the stressed plates relative to the colony numbers of control appearing on the non-stressed plates(Soto et al.1999).The survival rates of transformed E.coli BL21cells carrying pET32a-OsLEA5and pET-32a were compared for the assay of OsLEA5gene stress tolerance.Assay for the protective effect of the OsLEA5proteinon LDH activityLactate dehydrogenase from rabbit muscle was purchased from Sigma(USA)and diluted in100mM sodium phos-phate buffer(pH6.0)to afinal concentration of0.1mg/ml following the manufacturer’s recommendations.For the thermal inactivation assay,2l l of water or stock solutions of buffer,BSA,or OsLEA5were added to20l l of LDHdiluted solution in a microfuge tube.The enzyme mixture was incubated at43°C.At the specified time,2l l of ali-quots were collected and then kept on ice for5min.In order to measure the protective effect of the OsLEA5 protein on LDH activity after freeze–thawing and drying, BSA or OsLEA5were added to equal volumes of LDH at mass ratios of2:1,5:1,or10:1(test protein:LDH).Both water and stock solutions of buffer were used as control. For the freeze–thawing inactivation assay,the enzyme mixture wasflash-frozen in liquid N2,left for1min and then thawed for10min at room temperature.This consti-tuted one freeze–thaw cycle and was repeated up tofive times.For the desiccation-induced inactivation assay,the enzyme mixture was dried at25°C in an air stream at 50%relative humidity(RH)for5h,and then rehydrated immediately to the original volume with water before incubation for10min at ambient temperature.To determine LDH activity,2l l of the enzyme mixture was added to1ml of the assay buffer(100mM sodium phosphate buffer,0.1mM NADH,and2mM pyruvate). The LDH activity was monitored by the absorbance at 340nm for1min due to the conversion of NADH into NAD at37°C.During the experiments,water was used as controls to evaluate the dilution effect.All values given were expressed as percentage of the rate of the reaction measured for untreated samples.All samples were assayed in triplicate.ResultsBioinformatic analysis of OsLEA5Motif search analysis using InterProScan revealed that the novel LEA-like protein from Oryza sativa cv.Zhonghua 11contains a‘‘LEA_2’’motif(PF03168,1.9e-17),which was classified as group5C(D-95family)according to Battaglia’s classification(Battaglia et al.2008).Hence, the gene was named as OsLEA5.The full-length open reading frame(ORF)of OsLEA5cDNA is456bp(Gen-Bank Accession No.JF776156)and encodes a putative cytoplasmic polypeptide of151amino acids,with a cal-culated molecular mass of16.5kDa and a theoretical pI of5.07.The OsLEA5polypeptide is rich in Leu(10%), Ser(8.6%),and Asp(8.6%),while Cys,Trp,and Gln residue contents are very low,which are2,1.3,and1.3%, respectively.The predicted amino acid sequence shows 50–62%sequence identity with other members of atypical group5C.In addition,the conserved domain‘‘LEA_2’’was found in position about44–140amino acid of all proteins analyzed(Fig.1).Except for the identical region, the highly conserved Lys and Ala residues concentrate in the N-terminal regions and Gly residues in C-terminal regions,whereas the highly conserved acidic(Asp,Asn, Glu)and Pro residues distribute throughout the entire polypeptide(Fig.1).This feature may be involved in the molecular basis of primary structure,and thereby affect the protein folding type and the formation of spatial conformation.All members of the group5C predicted by Psipred’s analysis showed the significant secondary structural feature in mature proteins,that is increased content of b-sheet and with only1–4a-helical domains(Fig.1),which is in contrast to the canonical LEA proteins rich in a-helix.The structural data indicate that most LEA proteins including SMP(group5A)and LEA_3(group5B)are natively unfolded in a random coil structure(Boucher et al.2010; Boudet et al.2006),but it is striking that all members of the group5C are predicted by PDISORDERs analysis to be folded.Moreover,the instability index of group5C range from5.84to33.71,whereas other groups of proteins are generally up to35above(Table2).Protein targeting pre-dictions revealed that OsLEA5does not have N-terminal signal sequences and transmembrane domains.Currently, there is little structural information available for the LEA proteins.Kyte and Doolittle hydropathy plot revealed that the predicted OsLEA5protein is strongly hydrophobic throughout the length of the protein with the exception of the C-terminal region,with the GRAVY value of0.020 (Kyte and Doolittle1982).This is in coincidence with other groupfive members such as CaLEA6,IbLEA14,and PjLEA3,in which C-terminal is more hydrophilic than N-terminal(George et al.2009;Kim et al.2005;Park et al. 2011).In silico predictions show that the hydrophobic regions(I,II,and III)are found in the conserved motif of OsLEA5(Fig.2).Selecting several representative proteins of each group,we compared the hydrophilic degree and amino acid content of the group5C including OsLEA5with the various groups of LEA proteins.Among the LEAs shown in Table2,the most striking differences can be seen in the GRAVY values,the group5proteins especially5C (-0.360to0.075)are on average more hydrophobic than the rest of LEA groups,which show a wide range of GRAVY values(-1.494to-0.914)but are altogether quite hydrophilic.Among all of the LEA proteins listed, the group5C,represented by OsLEA5contains the lowest proportion of polar(hydrophilic)and small residues but the highest proportion of non-polar residues,whereas the content of the hydroxyl residues is little difference between groups.Groups5A and5C have their polar residues dis-tributed throughout the polypeptide,whereas group5B have more polar residues localized in the C-terminal half than the N-terminal half of the polypeptide(Fig.3).These features most likely contribute to hydrophobic character of the group5C proteins.The phylogenetic relationship between related group 5LEA proteins isolated from different plants was analyzed.An unrooted dendrogram showing clustering of OsLEA5with other group 5proteins was constructed (Fig.4).Group 5was easily separated into three distinct subgroups and thus suggests that the evolution of group 5proteins may come from various origins.OsLEA5is most closely related to the maize LOC100274480.In conclusion,rice OsLEA5belongs to the atypical group of hydrophobic LEA proteins,group 5C,which is significantly different from typical LEA proteins and even other members of group 5.The expression pattern of OsLEA5in riceIn order to well understand the function of this protein,the expression pattern of OsLEA5in rice was analyzed.Real-time PCR analysis showed that OsLEA5transcript was detected in the roots and leaves of rice plant at the seedling stage,their roots,stems,and leaves at the tillering stage,and their roots,stems,leaves,and panicles at the heading stage,filling stage,and full ripe stage,which demonstrated OsLEA5can express in different tissue organs during different development stages of rice (Fig.5).However,Fig.1Multiple sequence alignment of OsLEA5with its homologs belonging to group 5C LEA proteins from various higher plants.Asterisk identical residues,colon highly conserved residues,dot weakly conserved residues,dash gaps introduced for optimal alignment.The letters in the consensus sequence are as follows:capital letter completely identical amino acid residues,lower case letter chemically similar amino acid residues.N,D and E acidic and amide amino acid residues,R,K and H basic amino acid residues,A and G small amino acid residues,P proline residues.The secondary structure prediction using DSSP and PSIPRED algorithm for group 5C LEA proteins are shown in the sequences.The amino acids predicted to adopt a -helix conformation is gray and underlined ,and extended/b -sheet conformation is gray and non-underlined .The middle conserved ‘‘LEA_2’’motif (PF03168)is boxed .The GenBank accession numbers of proteins used for analysis in Online Resource 1significant differences were observed.The expression of OsLEA5in leaves at all times kept at a relatively higher level during the different development stages of rice.In both roots and panicles of full ripe stage,the expression levels of OsLEA5were dramatically upregulated,which were about17-fold and15-fold higher than those in leaves, respectively(Fig.5).These results illustrated that OsLEA5 might cope with various environmental stresses,matura-tion,and desiccation phases of rice seed development.Induction and SDS-PAGE analysis of fusion proteinin recombinant E.coliThe E.coli BL21transformants harboring the pET32a-OsLEA5and pET-32a were induced by0.8mM IPTG at 37°C for4h,respectively.The supernatants of sonicated lysates from bacterial cell were investigated to examine the presence of fusion protein by electrophoresing on SDS-polyacrylamide gels.According to the construction ofTable2Comparison of amino acid content(mol%),GRAVY and instability indexLEA group ResiduetypeSmall Non-polar ImionacidAcid?amide Basic Hydroxyl Hydrophilic GRAVY Instabilityindex ResiduegroupsAla,GlyIle,Leu,Met,ValPro Asn,sp,Gln,GluArg,Lys,HisSer,Thr Acid?amide?basic?hydroxyl5C OsLEA511.928.57.316.611.315.243.00.02023.54 BcLEA-29.832.0 5.918.312.414.445.10.01633.71 pcC27-4514.632.5 6.618.513.29.941.70.0759.25 Lemmi911.530.5 6.921.312.611.545.4-0.15726.47 ER510.030.0 6.920.612.513.146.3-0.14322.18 CaLEA610.430.5 6.722.011.613.447.0-0.19226.21 Lea14-A11.329.8 6.618.511.314.644.40.03219.30 D95-411.229.67.917.811.215.844.7-0.02119.37 GmLEA-213.126.9 6.422.114.110.646.8-0.28917.56 PcLEA1410.628.7 6.018.712.714.646.0-0.025 5.84 5A MtPM2526.221.2 5.822.38.814.245.4-0.26738.22 ECP3127.719.9 5.521.98.613.343.8-0.27539.82 PAP14026.120.2 5.022.610.511.444.5-0.26640.09 5B GhLea521.018.0 4.013.014.018.045.0-0.29349.10 SAG2122.619.6 3.116.614.417.548.5-0.36041.83 ATDI2122.221.1 3.814.513.518.246.2-0.25443.73 1TaEm25.511.7 1.125.517.016.058.5-1.30037.34 AhLEA1-223.412.8 1.129.818.112.760.6-1.37745.85 EMB56425.312.1 1.124.218.716.559.4-1.26541.65 2Dhn123.612.1 3.219.119.717.856.7-1.23421.12 GmPM1223.513.2 5.419.217.418.655.2-0.97134.10 ERD109.614.0 6.330.623.313.367.2-1.49463.21 3HVA130.27.40.024.815.820.360.9-1.03214.76 DcECP6326.511.30.726.820.69.256.6-1.14720.91 AtECP6322.713.7 1.125.019.913.658.5-1.02312.78 4GmPM2917.315.07.521.122.611.354.9-1.14050.15 BhLEA230.38.5 5.427.217.98.553.6-1.10931.50 PAP26019.414.89.018.623.99.752.2-1.04263.70 6PvLEA1820.99.3 4.723.318.617.459.3-1.35547.13 AhLEA8-118.110.77.428.815.016.059.8-1.38156.48 GmPM3521.111.7 6.326.412.717.957.0-1.11254.59 7Asr120.013.9 1.720.933.9 4.359.1-1.17439.49 lp3-322.413.9 3.224.521.28.554.2-0.91448.05 LLA2319.011.2 1.424.625.49.859.8-1.22735.45Similar residues are combined for simplicity,The GenBank。
干旱胁迫下的小麦LEA蛋白组学表达模式分析干旱是小麦生长过程中常见的一种环境胁迫因素，能够导致小麦生长发育受阻，产量严重降低。
为了适应干旱环境，小麦在干旱胁迫下会调节一系列生理和生化过程，其中包括低温胁迫相关蛋白（LEA蛋白）的表达。
LEA蛋白通常是非酶蛋白，富含多种氨基酸如谷氨酸、丙氨酸、半胱氨酸和亮氨酸等，能够在干旱胁迫下稳定蛋白结构并保护细胞内稀有的蛋白质。
为了研究干旱胁迫下小麦LEA蛋白的表达模式，研究人员通常采用基因组学和蛋白组学的方法进行分析。
基因组学分析可以通过测定小麦基因组中LEA蛋白基因的表达情况来了解其在干旱胁迫下的转录水平变化。
这可以通过RNA测序技术和实时荧光定量PCR等方法来实现。
蛋白组学分析则可以通过检测小麦LEA蛋白在干旱胁迫下的蛋白质水平变化来了解其在翻译水平上的调节机制。
这可以通过二维凝胶电泳、质谱分析和Western blot等技术来实现。
通过基因组学和蛋白组学的综合分析，可以得到干旱胁迫下小麦LEA蛋白的表达模式。
研究表明，在干旱胁迫下，小麦LEA蛋白的转录水平和蛋白质水平均有所上调。
这表明小麦在干旱胁迫下通过增加LEA蛋白的表达来增强其抵抗干旱胁迫的能力。
研究还发现小麦LEA蛋白的表达模式在不同干旱胁迫程度和时间点上可能存在差异。
在早期的干旱胁迫下，小麦可能通过增加LEA蛋白来维持细胞膜的稳定性和细胞结构的完整性。
而在长时间的干旱胁迫下，小麦可能通过延缓蛋白质降解和增加抗氧化酶的活性来提高LEA蛋白的表达。
干旱胁迫下的小麦LEA蛋白组学表达模式分析揭示了小麦在干旱胁迫下通过调节LEA蛋白的表达来适应干旱环境。
这些研究结果对于揭示小麦干旱适应机制、筛选耐旱相关基因以及培育耐旱小麦品种具有重要意义。
第9卷第4期2006年10月西安文理学院学报:自然科学版Journal of Xi .an University of Arts &Science(Nat Sci Ed)Vol.9 No.4Oct.2006文章编号:1008-5564(2006)04-0027-04收稿日期:2006-06-20基金项目:国家转基因植物研究与产业化开发项目(J2002-B-001).作者简介:赵咏梅(1968) ),女,陕西西安人,西安文理学院生命科学系副教授.第3组LEA 蛋白及其基因研究进展赵咏梅1,杨建雄2,俞嘉宁2(1.西安文理学院生命科学系,陕西西安710065;2.陕西师范大学生命科学学院,陕西西安710062)摘 要:胚胎晚期丰富蛋白(L EA 蛋白)是一种脱水保护剂,能够在干旱胁迫条件下保护生物大分子,尤其是膜结构的稳定性,减轻干旱造成的伤害.对其研究主要集中在植物胚胎学以及逆境生理学的领域.综述了有关第三组LEA 蛋白及其基因的近年研究进展,这些基因的表达或蛋白累积与植物的渗透胁迫抗性呈正相关,第3组LEA 基因可能具有的干旱保护功能已初步证实,同时分析了其广泛的应用前景.关键词:第3组LEA 蛋白;耐逆性;研究进展中图分类号:Q343.1 文献标识码:ALEA 蛋白(Late -embryogenesis -abundant protein)即胚胎发育晚期丰富蛋白,是指胚胎发生后期种子中大量积累的一系列蛋白质,最早发现于棉花胚胎发育后期的子叶.1981年Dure 等在胚胎发育后期的棉花子叶中分离到了一组丰富的mRNA,命名为Lea mRNA,其翻译产物即为LEA 蛋白.后来,许多LEA 蛋白或它们的基因在不同的植物包括大麦、小麦、水稻、玉米、棉花、葡萄种子和大豆中被发现[1].LEA 蛋白具有很强的亲水性和热稳定性,能够在水分亏缺时保护细胞膜系统及其它生物大分子免受破坏,同时,LEA 蛋白还受到发育阶段、脱落酸(ABA)和脱水信号的调节[2].LEA 蛋白是一类亲水性大家族,根据LEA 蛋白的氨基酸序列同源性比较结果,可将其分为6组,其中第3组LEA 蛋白成为当前植物胚胎学与逆境生理学的研究热点,受到广泛重视.1 LEA 蛋白的定位及性质LEA 蛋白是一类低分子量蛋白(大部分M r 在10~30kD,少部分在30kD 以上),广泛存在于高等植物中,一般认为,LEA 蛋白存在于植物的不同组织中,主要存在于细胞质中,尤其在胚细胞的细胞质中具有很高的浓度.例如在成熟的棉花胚细胞中,第3组LEA 蛋白占非细胞器胞质蛋白质的4%(约0.34mM ).最近也发现有定位于细胞核中的.Jose 等[3]通过免疫化学分析表明豆类幼苗中LEA 蛋白Pvlea 18定位在所有细胞的细胞核和细胞质区域,水分胁迫下,该蛋白在柱形组织和导管中,特别是在初生木质部细胞和根分生组织中大量积累.LEA 蛋白最重要的物理化学特性是具有很高的亲水性和热稳定性,即使在煮沸条件下也能保持水溶状态.研究表明,植物在水分胁迫时面临的最主要的问题是细胞组成成份的晶体化,这将破坏细胞的有序结构,而LEA 蛋白具有高度的亲水性,能把足够的水分捕获到细胞内,从而保护细胞免受水分胁迫的伤害.Baker 等[4]认为,LEA 蛋白出现在胚发育的晚期,积累始于成熟期末、干燥期启动之时,在以后干燥过程中含量达到高峰,种子萌发早期迅速消失.LEA 蛋白的另一个特点是,它们中的有些成员可被28西安文理学院学报:自然科学版第9卷ABA和水分胁迫因子诱导产生,有时二者的作用可相互代替.如:未发育成熟的小麦胚在体外培养时并不累积LEA蛋白,但若在培养基中加入ABA或施加渗透胁迫条件,则可诱导LEA蛋白的产生.Bray[5]用外源ABA胁迫或低温处理番茄离体胚和根、茎、叶等营养组织,能诱导到出特异LEA mRNAs和LEA蛋白.这表明,LEA蛋白虽是阶段发育专一的,但可以被诱导,且无组织专一性.2第3组LEA蛋白的结构LEA蛋白的共同特点是由极性氨基酸组成,为亲水性的多肽,甘氨酸含量>6%,亲水指数> 1.0[6],大多数LEA蛋白缺乏半胱氨酸和酪氨酸残基,但富含赖氨酸和甘氨酸.根据LEA蛋白氨基酸序列同源性和特定的基元序列,LEA蛋白被分为6组,即:D19LEA(第1组)、D11LEA(第2组,又称脱水素)、D7LEA(第3组)、D113LEA(第4组)、D29LEA(第5组)、D95LEA(第6组).由于各组LEA 蛋白序列和可能的蛋白质结构不同,每一组LEA蛋白在水分胁迫下有不同的功能.第3组LEA蛋白通常含有多拷贝的11个氨基酸组成的基元序列(TAQAAKEKAGE),孙海丹等[7]分析了8种重要农作物第三组LEA蛋白质序列中的94个11-氨基酸基序,得出LEA3蛋白质中11-氨基酸基序呈多拷贝串联排列,其长度占蛋白质序列总长度的40%~60%.带电荷/极性氨基酸和疏水氨基酸数目分别占重复序列氨基酸总数的70.89%和29.11%.第3组LEA蛋白质全序列平均亲水指数为-0.95~- 1.22,无信号肽序列.这11个氨基酸残基组成的基元序列可形成兼性A-螺旋结构,其中第1,2,4,5,9位疏水氨基酸形成螺旋蛋白质分子的疏水界面,在植物脱水时提供一个具疏水条区的亲水表面,处在亲水表面的带电基团可鳌合细胞脱水过程中浓缩的离子,如N a+和PO43-,其余位置的氨基酸形成螺旋分子的亲水界面.螺旋的疏水面可形成同型二聚体,同型二聚体随着外部离子浓度增强而改变其方向.此外,组成该蛋白的大多数氨基酸残基为碱性、亲水性氨基酸,无半胱氨酸和色氨酸,这些高电荷的氨基酸残基可以重新定向细胞内的水分子,束缚盐离子,从而避免干旱胁迫时细胞内高浓度离子的累积所引起的损伤,同时也可防止组织过度脱水[8].许多学者认为,LEA蛋白的亲水性也与其空间结构有关.提出一种假说认为第3组LEA蛋白含有的11个氨基酸的重复基元序列可形成氨基酸盐桥,从而充实蛋白质结构,提高细胞液中的离子强度,抵抗或减轻干旱形成的伤害.这类重复元件多数存在于兼性A-螺旋结构中,疏水和亲水氨基酸位于A-螺旋中的特定部位,在其内部可能形成维束管,表层有结合阴阳离子的功能,在水分缺乏时,此种蛋白在细胞中像可溶性糖一样,有束缚水分子的作用.研究发现,该组LEA蛋白大小差异很大,所含基元序列的拷贝数少的只有5个,如棉花LEA D7蛋白;多的则有几十个,如油菜LEA76蛋白有13个重复基元数[8],大豆cDNA pGm PM8和pGmPM10编码蛋白含有超过30个相连的基元序列[1].该组蛋白可依其基元序列的羧基端有无特定的氨基酸延伸区段可分为两种类型,一类是11个氨基酸组成的重复基元序列被与之相似的序列所分离,称第3组LEA 蛋白(I);另一类是在羧基末端被特定氨基酸延伸区段的出现而分隔,称第3组LEA蛋白(Ò)[9].大麦的HVA1与小麦的PMA2005在核苷酸水平和氨基酸水平上均有很高的同源性,分别达91%和95%,这两个单子叶植物的基因与胡萝卜的DC3和棉花的D7关系很密切,均属于第3组LEA蛋白(I);小麦的PMA1949,胡萝卜DC8和大豆的pGmPM2等属于第3组LEA蛋白(Ò)[10].对该族蛋白的研究比较多,如用多克隆抗体在小麦中检测到了27~30.5kD的4个该族蛋白,在玉米胚胎中也检测到了29kD的该族蛋白.其他的第3组LEA蛋白有小麦的PMA1959基因、WRAB19基因、TaLEA基因,大麦pH Va1基因、HAV22基因编码的蛋白质,葡萄种子的Plea76基因编码的蛋白及麝香百合M p2基因编码的蛋白.3第3组LEA蛋白的耐逆性植物严重脱水受害是由于水分减少使细胞结构凝结、细胞膜受损的缘故,而LEA蛋白则能削弱这种伤害.在干旱环境下,多数植物能诱导LEA蛋白的产生,虽然LEA蛋白的抗旱机制还不清楚,但大量试验已能够证实第3组LEA蛋白抗旱功能的真实性.例如,在严重干旱的小麦幼苗中,第3组LEA蛋白累积量的大小与组织的耐旱能力密切相关.此外,脱水还可造成第3组LEA mRNA 转录水平的增加[5].Xu 等将HAV1cDNA 全序列导入水稻,获得了抗旱、抗盐的转基因水稻,将其导入小麦,转基因小麦的生物量及水分利用效率增加,转入燕麦,转基因植物也表现为耐旱性提高[13].这些实验已初步证实了第3组LEA 基因可能具有干旱保护功能.近年来,俞嘉宁等利用同源序列扩增法从干旱48h 的小麦中克隆了3个第3组LEA 基因T aLEA 1、TaLEA 2、TaL EA 3,初步研究表明该组基因可能具有耐渗透胁迫的作用.用TaL EA 3做探针,对渗透胁迫下不同耐旱性的一对同核异质小麦幼苗进行Northern 杂交分析,表明在26.2%PEG6000胁迫处理3h 就可诱导TaL EA 3表达,6~12h 达到最高表达量,ABA 、NaCl 、冷处理均可诱导该基因表达,TaLEA 3基因的表达与所测品种的耐旱性呈正相关[12].TaLEA 3还能显著提高转基因酵母在渗透胁迫下和冰冻胁迫下的生长状况.对T aLEA 2基因在冷、干旱、ABA 、NaCl 处理下的表达模式分析,表明其表达可被冷胁迫诱导,对其他胁迫处理也有响应,TaL EA 2还能有效改善转基因酵母在山梨醇胁迫下的生长情况,提高转基因酵母在冰冻胁迫时的存活率.同时,研究发现,T aLEA 3与TaLEA 2有很高的同源性,TaL EA 2编码序列与小麦的WRAB19蛋白、WRAB17蛋白分别有84.4%和39.8%的同源性,与大麦中受冷胁迫的ES2A 也有39%的氨基酸同源性,而WRAB19蛋白与小麦受冷胁迫诱导有关,其也属于第3组LEA 蛋白.TaLEA 2基因编码一个由212个氨基酸组成的酸性,亲水性的蛋白,该蛋白具有5个11个氨基酸组成的重复序列.赵咏梅等[13]将TaL EA 2基因转入拟南芥中,对其功能作了进一步的研究,抗性实验表明,在0.8%的NaCl 胁迫下,转基因拟南芥的长势明显优于对照种;在10%,15%的PEG4000胁迫下,转基因拟南芥的生长状态明显改善,说明在一定条件下,转基因拟南芥的耐旱性及耐盐性均有所提高.这项研究为支持第3组LEA 基因及其LEA 蛋白在植物抗水分和盐胁迫时起保护作用的假说提供了直接的证据.4 研究展望第3组LEA 蛋白不仅可增强植物抵御干旱胁迫的能力,而且低温、盐胁迫也可使第3组LEA 基因表达及LEA 蛋白积累.如,小球藻C -27品系在冷锻炼过程中可产生低温诱导蛋白H iC6,它与甘蓝型油菜LEA76(一种LEA3蛋白)高度相似,桑树受冷害刺激后也可产生具12个11-氨基酸重复序列的蛋白质WAP27[7],这些研究表明,第3组LEA 蛋白很可能在多方面发挥作用.总之,随着人们对第3组LEA 蛋白功能的不断深入了解,人类将有可能更合理,更有效地利用抗逆LEA 基因,在抗逆分子育种的研究中取得突破性进展.[参 考 文 献][1] SWIR E CL ARK G A ,M ARCOT LE WR J.T he w heat LEA protein Em funct ions as an osmoprotective molecule insacharomyces cerevisiae[J].Plant M ol Biol,1999,39(1),117-128.[2] 张林生,赵文明.L EA 蛋白于植物的抗旱性[J].植物生理学通讯,2003,39(1):61-66.[3] 孙立平,李德全.L EA 蛋白的分子生物学研究进展[J].生物技术通报,2003,综述与专论(6):6-13.[4] 俞嘉宁,山 仑.L EA 蛋白与植物的抗旱性[J].生物工程进展,2002,22(2):10-14.[5] BRAY EA.M olecular respo nses to w ater deficit[J].Plant Physio l,1993,103:1035-1040.[6] CA RA Y ARR OYO A,COL M EN ER O JM ,GAR OIARR UBIO A ,et al .Highly hydrophilic proteins in prokaryotes andeukar yotes are common during conditions of water deficit[J].Biol Chem,2000,275(8):5668-5674.[7] 孙海丹,兰英,刘昀,等.L EA 蛋白质11-氨基酸基序与植物抗旱性[J].东北师范大学学报:自然科学版,2004,36(3):85-90.[8] DU RE L Ó.A repeat ing 11-mersamino acid motif and plant desiccation[J].Plant J,1993,363-369.[9] CU RR Y J,WAL T ER-SIM M ON S M K.U nusual sequence o f g roup 3L EA (Ò)M r na inducible by dehydration str essin wheat[J].Plant M olecular Biology,1993,21(5),907-912.[10] I NGRA M J,BART EL SD.T he molecular basis of dehydr atio n toler ance in plants[J].Annu Rev P lant Physiol Plant M ol Biol,1996,47,377-403.29 第4期赵咏梅,等:第3组LEA 蛋白及其基因研究进展30西安文理学院学报:自然科学版第9卷[11]XU D,DU AN X,WAN G B,et al.Ex pression of a late embr yogenesis abundant protein gene,HVA1,from barley con-fers tolerance to w ater deficit and salt stress in transg enic rice[J].P lant P hysiol,1996,110,249-257.[12]俞嘉宁,张林生,张劲松.小麦耐逆基因T aL EA3的克隆及在酵母中的功能分析[J].生物工程学报,2004,26(6),832-838.[13]赵咏梅,杨建雄,俞嘉宁,等.小麦耐逆基因-T aLEA2转化拟南芥的研究[J].西北植物学报,2006,26(1):1-6.[责任编辑马云彤]Research Progress in Group3LEA Protein and GeneZHAO Yong-mei1,YANG Jian-x iong2,YU Jia-ning2(1.Department of L ife Science,Xi.an U niversity of Arts and Science,Xi.an710065,China;2.College of Life Science,Shaanxi Normal U niversit y,Xi.an710062,China)Abstract:Late-embryog enesis-abundant protein(LEA protein)is a kind of protective agent of dehydration, w hich can protect biological macromolecules from the w ater-stress of environment,in particular,maintain the cell mem brane stability,and mitigate harms from drought.Study of LEA proteins w as one of the hot fields in plant embryolog y and stress physiology.The recent research progress of Group3LEA protein and g ene research w as summarized.It show ed that these gene expressions or protein accumulation had positive correlation w ith the botanical osmotic pressure,and it w as confirmed that Group3LEA gene m ight resist droughts.Prospects of w ide application w ere also analyzed.Key words:Group3LEA protein;stress tolerance;research progress。
干旱胁迫下的小麦LEA蛋白组学表达模式分析小麦是世界上最重要的粮食作物之一，然而干旱胁迫是影响小麦产量和品质的重要因素。
为了适应干旱环境，植物会通过多种途径调节其基因表达和代谢途径。
低温和低渗透胁迫下的蛋白组学研究已经证明了LEA（Late Embryogenesis Abundant）蛋白在植物抗逆过程中的重要作用。
关于干旱胁迫下的小麦LEA蛋白组学表达模式的研究相对较少。
本文将对干旱胁迫下的小麦LEA蛋白组学表达模式进行分析，以期为小麦抗逆机制的研究提供参考。
一、干旱胁迫对小麦生长发育的影响干旱胁迫是世界范围内最常见的生物胁迫之一，对农作物的生长发育和产量具有极大的影响。
小麦作为主要的粮食作物之一，其叶片、茎和根系都会受到干旱胁迫的影响。
在受到干旱胁迫的情况下，小麦植株的生长速度减慢，叶片出现卷曲、枯黄等症状，根系的生长也会受到严重抑制。
干旱胁迫会触发植物体内一系列的适应性反应，包括调节水分利用效率、调节渗透调节物的积累和分布、以及调节植物内部的抗氧化系统等。
LEA蛋白作为植物在应对胁迫时的一种重要蛋白，其在干旱胁迫下的表达模式对小麦的抗逆能力具有重要的意义。
二、小麦LEA蛋白的分类和功能LEA蛋白是一类具有保护作用的蛋白，在植物的干旱胁迫中扮演着重要的角色。
LEA蛋白可以分为不同亚组，包括LEAⅠ、LEAⅡ、SMP和dehydrin等亚组。
这些LEA蛋白在植物的耐旱、耐寒、耐高渗透胁迫等逆境中均发挥着重要的作用。
LEA蛋白通过保持细胞内部的稳定性和蛋白的活性，促进细胞膜的稳定性，维持细胞内水分平衡等多种方式来帮助植物应对胁迫环境。
LEA蛋白在植物的抗逆过程中具有重要的生物学功能。
近年来，随着蛋白质组学技术的不断发展，研究人员对植物在胁迫条件下的蛋白组表达模式进行了系统研究。
干旱胁迫下的小麦LEA蛋白组学表达模式也成为了研究的焦点之一。
研究表明，干旱胁迫会导致小麦体内多种LEA蛋白的表达水平发生变化。
LEA蛋白及其在作物抗逆过程中的作用王梦飞;滑璐玢【摘要】干旱、寒冷、盐碱等是作物在生长发育过程中常见的逆境因子,作物的生长发育和产量都会受这些逆境因子的影响,所以越来越多的研究者热衷于作物抵抗逆境胁迫的研究.LEA蛋白（胚胎发育晚期丰富蛋白）属于逆境胁迫响应蛋白,广泛存在于植物中.文章主要对LEA蛋白的分布、分类和功能、LEA蛋白在作物抗逆过程中的研究进展做简要综述.【期刊名称】《北方农业学报》【年(卷),期】2018(046)004【总页数】7页(P70-76)【关键词】LEA蛋白;分类;功能;作物抗逆性【作者】王梦飞;滑璐玢【作者单位】[1]山西省农业科学院高寒区作物研究所,山西大同037008;;[2]内蒙古博物院,内蒙古呼和浩特010000;【正文语种】中文【中图分类】Q51作物在整个生命周期中不可避免地会受到干旱、盐碱等逆境因子的胁迫，这些逆境因子会对其生长、发育以及产量造成严重的影响。
作物为抵御这些不利的外界环境，在长期的进化过程中形成了一些特定、复杂的防御机制。
这些防御机制大致可以分为3类：第1类是参与非生物胁迫转录调控和信号转导的转录因子、蛋白激酶和蛋白磷酸酶，属于调节性蛋白相关基因；第2类是在植物受胁迫过程中吸收、转运水分和离子的水孔蛋白和离子转运体；第3类是可以直接参与胁迫响应的各类功能性蛋白[1]。
LEA蛋白（Late embryogenesis abundant protein）即胚胎发育晚期丰富蛋白，是一类参与植物抵御逆境胁迫的功能性蛋白，主要作用是保护遭遇逆境的植物体能够维持正常的生命代谢。
1 LEA蛋白简介1981年，研究人员在棉花（Gossypium spp.）的子叶中发现了一种蛋白，其特征是能够在棉花种子脱水成熟期大量积累，并保护组织细胞在种子成熟过程中免受脱水给种子造成的伤害，因此该蛋白被命名为胚胎发育晚期丰富蛋白[2]。
在棉花中发现LEA蛋白后，研究人员在小麦（Triticum aestivum L.）、大豆（Glycine max L.）和玉米（Zea mays L.）等作物中也相继发现了LEA蛋白的存在[3]。
LEA蛋白及其在植物抗逆改良中的应用王艳蓉;张治国;吴金霞【摘要】干旱、盐碱、高温和寒冷等逆境制约着植物的生长发育。
植物中含有一组亲水性极强的蛋白，称为胚胎发育晚期丰富蛋白（late embryogenesis abundant，LEA蛋白），在自然条件下这种蛋白质一般在种子发育晚期积累，其对多种非生物胁迫具有很强的抵抗能力，并能响应干旱、寒冷、高盐和ABA等信号。
LEA蛋白通过保持细胞渗透压、保护细胞膜结构、作为分子伴侣保护其他蛋白等方式维持植物正常的代谢反应。
就LEA蛋白的分类、结构、抗逆机制以及在植物抗逆改良中的应用进行了简要综述。
%Adverse conditions, including drought, salinity and extreme temperatures, often restrict the growth and development of plants. In plants, there exist a group of highly hydrophilic proteins, known as LEA(late embryogenesis abundant)proteins, which generally accumulate at the last stage of embryogenesis under natural conditions. It has a strong resistance to various abiotic stresses, and can respond to drought, cold, high salt and ABA signals. LEA proteins can maintain normal metabolic reactions of plants by maintaining cellular osmotic pressure, protecting the cell membrane structure and functioning as a molecular chaperone to protect other proteins. In this paper, the classification, structure, stress tolerance mechanisms of LEA protein and its application in improvement of stress tolerance in plant were reviewed.【期刊名称】《生物技术通报》【年(卷),期】2015(000)003【总页数】9页(P1-9)【关键词】LEA蛋白;非生物胁迫;植物抗逆性【作者】王艳蓉;张治国;吴金霞【作者单位】兰州大学草地农业科技学院，兰州 730000;中国农业科学院生物技术研究所，北京 100081;中国农业科学院生物技术研究所，北京 100081【正文语种】中文植物所在环境常常会对其生存产生各种不利的影响，这些不利的环境因素统称为逆境。
25卷2期Vol.25.No.2草业科学PRAT ACULTURAL SCI ENCE972/2008植物LEA蛋白及其基因家族成员PM16研究进展王康,朱慧森,董宽虎(山西农业大学动物科技学院,太谷山西030801)摘要:通常在逆境胁迫下植物细胞会积累一系列的蛋白质来保护细胞免受伤害,其中LEA蛋白是目前研究最为普遍的一种。
综述了与抗旱性密切相关的LEA蛋白的结构、性质、分类与功能,并对一经在Genbank 中登录的LEA基因家族成员进行了汇集整理。
以大豆Glycine ma x LEA蛋白基因家族成员之一的P M16为代表,对其基因以及所编码的LEA蛋白进行了生物信息学分析。
关键词:抗旱性;LEA蛋白;LEA基因;PM16中图分类号:Q943文献标识码:A文章编号:100120629(2008)022*******在农业生产中,经常会遇到各种不良的环境条件,如干旱、洪涝、低温、高温、盐渍化以及病虫害等,世界上每年都有不同程度的自然灾害发生。
陆生植物最常遭受的环境胁迫就是缺水,植物在复杂多变的环境生长过程中,突发性和短暂性干旱经常发生,从而影响着植物的正常生长发育,同时当植物受到冷害和盐胁迫时也会产生由于细胞水分亏缺引起的生理干旱。
我国西北、华北地区干旱缺水是影响农林生产的重要因素,南方各省虽然雨量充沛,但由于各月分布不均匀,也时常有干旱危害。
因此植物的耐旱性分子机理研究及其应用成为当前研究的一个热点。
迄今为止,国内外科研工作者已经形成一种共识[1],就是通过筛选与植物的抗旱性相关的基因,认识其功能,揭示抗旱相关因子的信号转导途径,在此基础上,通过生物技术手段进行转基因育种等,从而获得耐旱能力强的新品种。
通常在逆境胁迫下,植物体内有些正常蛋白质的合成被抑制,但同时也会诱导出许多新蛋白质的合成,即在逆境中出现一些起保护细胞作用的逆境蛋白[2,3],如热休克蛋白(heat shock pro2 teins,H SPs)、低温诱导蛋白(low2temperatur e2 induced protein)、病原相关蛋白(pathogenesis2 related proteins,PRs)等。
