LEA蛋白研究进展

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植物低温胁迫适应性应答综述

植物低温胁迫适应性应答综述摘要：对植物低温胁迫适应性应答的研究进展，包括低温诱导蛋白、低温转录因子、低温信号转导、不饱和脂肪酸酶，以及低温次级氧胁迫进行了综述。

关键词：植物；低温胁迫；适应应答低温胁迫包括0-12℃之间的冷胁迫(chillingstress)和0℃以下的冰冻胁迫(freezing stress)两种。

它是一种严重的自然灾害，不仅限制作物的区域分布和生存，还对作物产量有很大影响。

探讨植物在低温胁迫下的生理生化变化及其抗寒冻机理。

对改善作物抗寒冻性能，提高经济作物产量，改善环境绿化状况均有十分重要的理论与经济意义和社会效益，是人们关注和研究解决的植物生理学和农业问题之一。

1 低温胁迫下的植物损伤环境温度改变会引起物质在水溶液中发生物理化学变化。

随着温度降低，水分子的粘滞性可以增大几倍。

使得溶剂以及水分子的扩散速率下降，盐的溶解性也降低，而气体的溶解性增大。

生物体缓冲系统的pH提高。

另外，细胞结冰往往伴随着脱水。

使细胞内渗透压增大，细胞体积缩小。

质膜系统和细胞骨架受到损伤，气体交换受阻，生物大分子结构改变并导致功能丧失，有害物质积累，植物细胞器如线粒体、叶绿体、核糖体的结构与功能也受到影响。

植物体内包括光合、呼吸、生长发育、代谢、蒸腾以及营养水分吸收等在内的几乎所有的生命活动都会不同程度地受到寒冷胁迫的干扰。

有关植物冷害的最早学说是Lyons在1973年提出的“膜脂相变”学说。

该学说认为，与热激胁迫所引起的蛋白质变性以及折叠受阻不同，低温对冷敏感植物的伤害首先是改变了磷脂双层膜的膜相，尤其是改变了质膜的空间构象和物理状态，使从片层(lamellar)转变为非片层(non-lamellar)或六方晶Ⅱ(hexagonalⅡ)，从液晶相转变为凝胶相。

膜相的改变可能抑制细胞膜发挥正常功能，而构象的改变影响了膜的稳定性，使蛋白质从膜上解聚下来，发生膜融合。

2低温胁迫对植物细胞生物学和生物化学的响应虽然植物不能像动物那样靠运动来趋利避害，但在长期进化过程中也形成了多种在寒冻环境下生存的适应机制，包括被动适应机制和主动适应机制。

leaa 氨基酸-概述说明以及解释

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。

LEA基因研究的经典文献——LEA基因综述(目前最全面深刻的LEA基因总结文章)

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。

LEA蛋白及其在作物抗逆过程中的作用

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]。

植物抗旱抗旱机理及其相关基因研究进展

植物抗旱抗旱机理及其相关基因研究进展植物抗旱是指植物在干旱等恶劣环境下，能够通过一系列适应性生理和生化机制来维持生长和发育的能力。

植物抗旱机理主要涉及到水分利用效率提高、减少蒸腾速率、促进根系发育和增强细胞膜的稳定性等方面。

近年来，随着基因测序技术的快速发展，植物抗旱相关基因的研究进展迅速。

植物的抗旱机制主要包括避免脱水、渐进脱水耐受和耐旱维持三个阶段。

避免脱水是指植物通过调节气孔的开闭来减少水分蒸腾，防止脱水。

渐进脱水耐受是指植物在长期干旱时，通过一系列适应性调节，逐渐适应干旱环境并维持正常生长和发育。

耐旱维持是指植物在长时间干旱条件下，能够维持细胞内水分平衡，避免细胞脱水，保持生长和发育活力。

植物抗旱的分子机制涉及到多个基因家族的调控。

其中，ABRE （Abscisic Acid-responsive Element）、DRE（Drought-responsive Element）和LEA（Late Embryogenesis Abundant）等基因家族被广泛研究。

ABRE基因家族与植物在胁迫条件下的ABA合成与信号传导过程中发挥重要作用，参与调控植物抗旱能力的提高。

DRE基因家族是植物耐旱途径基础基因，参与调控植物在水分胁迫下的抗逆应答。

LEA基因家族的蛋白质在干旱逆境下的活化与折叠起到了关键作用，参与细胞质和叶绿体中蛋白质合成抗旱蛋白并降低脱水损伤。

除了以上基因家族，研究还发现其他抗旱相关基因，如水通道蛋白基因、抗旱酶基因、氮代谢酶基因等。

水通道蛋白基因能够调节植物细胞水分传输，提高植物的抗旱能力。

抗旱酶基因参与植物在干旱逆境下的生理代谢过程，保护细胞膜的完整性和功能。

氮代谢酶基因在植物受到干旱胁迫时能够促进植物根系的发育，增加植物对水分的吸收能力。

基因研究的进展有助于提高植物的抗旱能力，并为植物育种和遗传改良提供了理论基础。

通过转基因技术，研究者可以将抗旱相关基因导入非耐旱植物中，提高其抗旱能力。

新一代大肠癌相关抗原LEA的研究进展

靶抗原。
参考文献
意 10 义11 2
[1) 张海林，施华芳，米竹青，云南省景洪市虫媒病调查分等. 析闭. 地方病通报， 2000,15(4):35-38. [2) 步恒富，吴庆丽，祝庆余，基孔肯亚病毒中和单克隆抗等.
的细胞表面可溶性肿瘤相关抗原，由内质网合成，并
作者单位:1. 南医科大学，广东，广州510515
2. 南医科大学生技术学院，广州510515 物 ‘ 广东，
趋接近。这表明，腺瘤在组织形态学上发生癌变之前，由于肿瘤基因的活化，已逐渐发生了细胞分子生物学方面的变化。因此，对肿瘤相关抗原 LEA 的检测，可能有助于判断腺瘤的恶变倾向。这一结果亦支持腺瘤一癌序贯论tln。因此， LEA有可能成为对大肠癌癌前病变早期诊断的有实用价值的肿瘤标志物。 2.2 LEA 作为新一代的肿瘤标志物目前研究得较多的大肠癌肿瘤标志物有 CA19- 9,CA242 和CEA 等，以下将对各种标志物的特点和临床应用价值作一个比较。 CEA:最早发现的结肠癌血清标志物。CEA 为一种酸性蛋白，胚胎期在小肠、肝脏、胰腺合成，成人血
noembr onic antiger ， y n 癌胚抗原)的大分子量细胞表面
糖蛋白抗原，其抗原决定簇的主要成分是唾液酸，相
对分子质量>500 kDao LEA 可能作为新型临床组织 >
组织化学ABC 法，对大肠腺瘤进行免疫组织化学染色，研究该抗体对应抗原LEA1 1的表达与腺瘤组织学 ',8 分类及异型程度的关系。研究结果表明，是对大 LEA 肠癌，特别是对高分化腺癌较特异的肿瘤相关抗原[曳 l 但LEA在大肠腺瘤中的表达状况，以及这种表达与大肠癌的关系，前尚未见国内目有系统的研究报道。本研究显示，在腺瘤中表达的阳性率明显高于炎性 LEA 息肉和正常a 膜; 在腺组织中，的表达随着腺瘤 LEA 的异型程度的增加而增强，重度异型腺瘤的表达阳性率及染色强度均明显高于轻度异型腺瘤，随着腺瘤细胞中绒毛成分的增多，的表达亦逐渐增强， LEA 其在管状腺瘤中的阳性率及染色强度明显低于绒毛状腺瘤。值得注意的是，无论是在绒毛状腺瘤或是重度异型腺瘤组织中，的表达阳性率均与大肠癌相似， LEA 提示腺瘤和癌组织中含有共同的肿瘤相关抗原 LEA,

《2024年ELAVL1通过增强TL1AmRNA稳定性促进胃癌细胞增殖和迁移》范文

《ELAVL1通过增强TL1A mRNA稳定性促进胃癌细胞增殖和迁移》篇一摘要：本文探讨了ELAVL1基因通过增强TL1A mRNA稳定性的作用机制，对胃癌细胞增殖和迁移的影响。

研究结果显示，ELAVL1的表达与胃癌细胞的生长和转移密切相关，揭示了其潜在的治疗靶点。

本文采用分子生物学技术、细胞生物学实验等方法，详细探讨了ELAVL1的作用机制及其对胃癌发展的影响。

一、引言胃癌是全球最常见的恶性肿瘤之一，其高发病率和死亡率已成为重要的公共卫生问题。

随着分子生物学的发展，基因在胃癌发生发展中的作用日益受到关注。

其中，ELAVL1（胚胎致死异常视蛋白样1）作为一种RNA结合蛋白，在多种癌症中发挥重要作用。

本研究旨在探讨ELAVL1通过增强TL1A mRNA稳定性对胃癌细胞增殖和迁移的影响。

二、材料与方法1. 材料：选用胃癌细胞系及正常胃黏膜细胞系，ELAVL1及TL1A的特异性引物、抗体等。

2. 方法：通过实时荧光定量PCR、Western blot等技术检测ELAVL1及TL1A的表达水平；采用RNA稳定性实验验证ELAVL1对TL1A mRNA稳定性的影响；利用细胞增殖和迁移实验探讨ELAVL1对胃癌细胞生物学行为的影响；并采用分子生物学技术，如荧光报告基因分析、siRNA干扰等，探究其作用机制。

三、实验结果1. ELAVL1与TL1A表达的关系：在胃癌细胞中，ELAVL1的表达与TL1A的表达呈正相关。

通过RNA稳定性实验发现，ELAVL1能够增强TL1A mRNA的稳定性。

2. ELAVL1对胃癌细胞增殖的影响：通过细胞增殖实验发现，ELAVL1的表达增加能够显著促进胃癌细胞的增殖。

3. ELAVL1对胃癌细胞迁移的影响：划痕实验和Transwell迁移实验显示，ELAVL1的高表达有助于胃癌细胞的迁移能力增强。

4. 作用机制探讨：通过荧光报告基因分析和siRNA干扰等技术，发现ELAVL1通过与TL1A mRNA结合，增强其稳定性，从而促进胃癌细胞的增殖和迁移。

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[ 34 ]
在干旱状态下都
形成了高级结构。 11 3 LEA 蛋白的亚细胞定位通过计算机软件 TMHMM v . 2
[ 35]
预测和试验研
。
究, 证明了 LEA 蛋白不是跨膜蛋白 , 而是定位在包括叶绿素、线粒体、细胞核和细胞质等亚细胞中 ( 表 1)。 [ 36] 研究报道 , N末端的信号肽证明了 3 组蛋白的两个密切联系的蛋白定位在内质网膜 ( ER )上。当信号肽被清除后该蛋白的含量也就急剧下降了。其他一些信号也可以控制蛋白定位在线粒体和质粒中。例如, 玉米中的第 2 组 LEA 蛋白 DHN1 /Rab17 的定位取决于该蛋白的丝氨酸是否磷酸化, 当去磷酸化后该蛋白就可以在细胞质之中定位。应用生物信息学的方法预测了一些 LEA 蛋白的定位 (表 2), 认为第 1 组 LEA 蛋白是定位于细胞核中, 而线虫中的第 3 组 LEA 蛋白均匀的分布在细胞质中
[ 15] [ 16] [ 13 , 14]
的 LEA 蛋白 EM B - 1 没有二级结构和三级结构。截形苜蓿 (M edicago truncula ta ) 中的第 1 组 LEA 蛋白 M tEm 6 的 50 % 没有蛋白结构 , 尽管有 37 % A 螺旋和 10 %是 B -折叠
收稿日期 : 2009 - 04 - 14 基金项目 : 国家科技 / 十一五 0支撑计划国家现代农业产业技术体系建设专项资金资助 , 中部温带地区优质高产、抗逆新品种选育的产业化示范作者简介 : 白永琴 ( 1985-) , 女 , 在读硕士研究生 , 从事牧草生物技术研究 ; E-m ai:l baiyongq -03@ 163 . com 通讯作者 : 杨青川, E -ma i:l qchyang66@ yahoo. com. cn , Te: l 010-62815996
2
生物技术通报 B iotechnology Bulletin
[ 10]
2009 年第 9期
列富含高比例的带电荷氨基酸和甘氨酸。第 2 组 LEA 蛋白也称为脱水素, 其一级结构特征: 在 C-末端或附近具有富含赖氨酸基元序列, 也称 K-片段, 在高等和低等植物中高度保守, 大约有 15 个氨基酸残基 ( EKKG I M DK I KEKLPG)组成 , 它的主要功能是作为分子伴侣。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 )
[ 13] [ 12] [ 11]
卷曲等结构。他们认为这种结构的形成是由于该氨基酸组成中富含甘氨酸 , 谷氨酸和谷氨酰胺残基等亲水基团造成的。这些是最早发现的 LEA 蛋白的典型特征。菜豆 ( P isu m sa tivum ) 中的第一组 LEA 蛋白 P11只有 2% 折叠成 A -螺旋 , 使得该蛋白在 80b C 水浴中也能处于溶解状态, 从而有利于脱水应激
研究表明该组蛋白比球蛋白能结合更多的水分。第 3 组 LEA 蛋白也是同样 , 如线虫 ( Aphelenchus avenae)
[ 27]
和大豆 ( soy bean )
[ 3 组 LEA 蛋白也可
组 LEA 蛋白的结构是 A -螺旋的 , 其实不然, 它们主要由球蛋白组成 , 在亲水力的作用下它们围绕亲 [ 30] 水核进行折叠 , 在结合了分子伴侣或者离子时也会进行非天然折叠
Key w ords :
L ate e m briogenesis abundant prote in G ene expression and regulation Env ironm enta l stress
晚期胚胎发生丰富蛋白 ( LEA) 是生物体中广泛存在的一类与渗透调节有关的家族蛋白。研究发现在植物胚胎发育晚期 , LEA 蛋白表达量十分丰富, 在成熟的棉花胚细胞中 , D7LEA 蛋白占非细胞器胞质蛋白质的 4 % (约 0134 mM ) , 而且在环境胁迫如干旱、低温、盐胁迫、 ABA、紫外辐射和 NaHCO3 等
[ 25 ] [ 26 ]
以及 Lea5( D- 73) 和 Lea14( D-
。
95) 。W ise 利用生物信息学 ( POPP ) 技术分析定义了 LEA 蛋白的超家族 ( SF s), 而每个主要组是由一个或多个亚组 SF s组成, 比如第 2组 LEA 蛋白又可以分为 2a和 2b
[ 17]
[ 24]
, 第 2 组 LEA 蛋白也有相似
的结构特征 , 比如在玉米 ( Z ea m ays ) , 复活植物 ( C raterostigm a p lantag ineum ), 柑橘 (C itrus ) 和拟南芥 (A rabidop sis ) 中具有非常显著的非折叠结构
[ 19 ] [ 18]
。环境条件的改变也可以改
变它们的折叠情况, 阔叶香蒲 ( T ypha latifo lia ) 中的第 3 组 LEA 蛋白 ( 8 kD ) 在快速干旱条件下形成高 [ 32 ] 度 A 螺旋 , 同样, 线虫的第 3 组 LEA 蛋白 Aav LEA1
[ 33]
和豌豆线粒体的 LEAM
生物技术通报
# 综述与专论 #
B IOTECHNOLOGY BULLETIN
2009 年第 9期
LEA 蛋白研究进展
白永琴
摘要:
杨青川
( 中国农业科学院北京畜牧兽医研究所 , 北京 100193)
LEA 蛋白 ( late embr iogenesis abundant prote in, LEA ) 是生物体中广泛存在的一类与渗透调节有关的家族蛋白 ,
该蛋白的编码基因在植物种子胚胎发育晚期表达量丰富 , 而且在环境胁迫如干旱、低温、盐胁迫、 A BA、紫外辐射和 N a HCO3 等条件下 LEA 基因的 mRNA 也会大量累积。 LEA 蛋白显著的理化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。 LEA 蛋白在细胞中可以稳定细胞膜结构 , 作为分子伴侣 , 具有结合离子和防止氧化等作用 , 被认为是在胁迫过程中对植物起保护作用的物质之一。针对这些重要特性 , 分别综述了 LEA 蛋白的分类、结构、编码基因和表达调节方式及其在植物生长过程中的作用。关键词 : LEA 蛋白基因表达和调节逆境胁迫
[ 1]
1 LEA 蛋白概述
11 1 LEA 蛋白的分类和特点 1981年 Dure 等在胚胎发育后期的棉花子叶中首先发现了 LEA 蛋白的存在。后来 , 在包括大麦、小麦、水稻、玉米、葡萄种子和大豆在内的 20 种高等植物中也发现了 LEA 蛋白。由于 LEA 基因序列的多 [ 7] [ 8] 样性 , 除了在高等植物体内, 在线虫 , 苔藓中也发现了 LEA 蛋白的存在, 使得 LEA 蛋白的分类从发现以来一直是充满分歧。最早, 根据它们普遍存在的特 [ 9] 殊氨基酸序列, LEA 蛋白被分为 3组。第 1 组 LEA 蛋白具有多拷贝串连的 20 个亲水氨基酸序列 , 该序
[ 38] [ 37]
通过凝胶法和斑点杂交法的限制性片段多态性 [ 20] 证明陆地棉 ( G. hirsu tum ) LEA 基因是单拷贝。插入外源基因的拷贝数低 ( 1 或 2 个 ) 能较好的表达, 插入的拷贝数多则会导致表达的不稳定甚至基因沉默现象。因此, 外源 LEA 基因可以较好的在转基因植物中表达。 11 2 LEA 蛋白的结构 1985 年, M cCubb in 等最早研究了小麦 ( T riticum aestivum ) 第 1 组 LEA 蛋白基因 Em 的结构特征, 发现该蛋白的结构比一般的球蛋白更疏松易变, 且处于无规则结构状态, 例如 , A -螺旋、 B折叠或者自由
[ 31]
以进行亚组分类, 但是它们的功能和结构特征与已知 LEA 蛋白的关联性不大。 LEA 蛋白相对分子质量大小各异 , 例如 , 阔叶香蒲 ( T ypha latif olia ) 的第 3组 LEA 蛋白是 8 kD, 而线虫 ( C. elegans )中的编码 Ce- lea - 1 的第 3 组 LEA 蛋白是 77 kD, 而且它们的等电点也不相同。其显著的理化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。 LEA 蛋白的高度亲水性有利于 LEA 蛋白在植物受到干旱失水时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
[ 21 , 22 ]
。
。同样, 也有报道胡萝卜 ( Daucus carota ) 中
[ 23 ]
Dure 等在 1993 年提出了根据棉花种子的原型进行 LEA 蛋白的命名方法, 例如, D- 19第 1组, D- 11第 2组, D- 7 第 3 组 , 但是后来研究者们都直接用第 1 组, 第 2组, 第 3组。尽管大多数 LEA 蛋白被分为 3 大类之中, 但是还有报道根据序列的相似性和特征序列分出了更小组别 : 第 4 组 ( D- 113), 第 5 组 ( D- 29) 和第 6组 ( D- 34)
erally invo lved in os m oregu lation . T hese genes encod ing prote ins are expressed abundantly at the late stage embryon ic developm ent o f plant seeds , or subjected to env ironm enta l stress such as drought , low temperature , salt stress, ABA, u ltrav io let radiation and sa line -al ka liza tion stress. Sign ificant phy sica l and chem ical prope rties o f LEA pro te in feature high hydrophilic and ther m al stab ility , even under condition o f bo iling w ate r they canm a inta in w ate r - solub le state . LEA prote ins can stab ilize the cellmembrane structure , and as mo lec ular chaperones , can comb ine ions and preven t ox ida tion . T he c lassifica tion and structure of LEA pro te in, encod ing genes , the ir ex pression and regulation and the ir ro les in different physio log ica l processes w ere summ a rized in th is paper .