Expression in Escherichia coli of Three Different Soybean Late Embryogenesis Abundant （LEA） Genes to Investigate Enhanced Stress Tolerance

In order to identify the function of late embryogenesis abundant (LEA) genes, in vitro functional analyses were performed using an Escherichia coli heterologous expression system. Three soybean late embryogenesis abundant (LEA) genes, PMll (GenBank accession No. AF004805; group 1), PM30(AF117884; group 3), and ZLDE-2 (AY351918; group 2), were cloned and expressed in a pET-28a system.The gene products were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. E. coli cells containing the recombinant plasmids or empty vector as controls were treated by salt and low temperature stress. Compared with control cells, the E. coli cells expressing either PMll or PM30 showed a shorter lag period and improved growth when transferred to LB (Luria-Bertani) liquid media containing 800 mmol/L NaC1 or 700 mmol/L KC1 or after 4℃ treatment. E. coli cells expressing ZLDE-2 did not show obvious growth improvement both in either high KC1 medium or after 4℃ treatment. The results indicate that the E. coli expression system is a simple, useful method to identify the functions of some stress-tolerant genes from plants.     

Expression of plant group 2 and group 3 lea genes in Saccharomyces cerevisiae revealed functional divergence among LEA proteins

To study functions of late embryogenesis abundant (LEA) proteins, which accumulate in plant cells under water deficit conditions, in vivo functional analyses were carried out using a yeast (Saccharomyces cerevisiae) heterologous expression system. Two lea genes, tomato le4 (group 2) and barley HVA1 (group 3), were expressed under the GAL1 promoter, and the gene products were detected using specific antisera. The growth of the transformants was scored and compared with a control strain to analyze the effect of these proteins on yeast cells under stress conditions. The yeast cells expressing HVA1 showed shorter lag period when transferred to a medium containing 1.2 M NaCl as compared to a control strain, while the cells expressing le4 did not show improved growth. Attenuated growth inhibition in a medium containing 1.2 M KCl was observed in the yeast cells expressing le4 and HVA1. No obvious growth improvement was observed in a high sorbitol medium in the cells expressing either le4 or HVA1. Increased freezing tolerance was observed in both lea-expressing cells, while no effect on heat tolerance was observed. These results support the hypothesis that different LEA proteins play a distinctive role in the protection against cellular dehydration.     

Two New Group 3 LEA Genes of Wheat and Their Functional Analysis in Yeast

The group 3 late embryogenesis abundant （LEA） proteins are thought to protect cells from stresses associated with dehydration during periods of water deficit. To investigate the functions of different members of the group 3 LEA genes, we isolated and characterized two new group 3 LEA genes, namely TaLEA2 and TaLEA3, from wheat （Triticum aestivum L.） and introduced TaLEA2 and TaLEA3 into Saccharmyces cerevisiae to examine the effect of these genes on yeast cell tolerance to osmotic, salt, and cold stresses. The TaLEA2 gene encoded a protein of 211 amino acids and possessed five repeats of 11-mer amino acid motifs. The TaLEA3 gene encoded a polypeptide of 211 amino acids with nine repeated units. Overexpression of TaLEA2 and TaLEA3 improved stress tolerance in transgenic yeast cells when cultured in medium containing sorbitol, salt and-20℃ freezing treatments respectively. However, the yeast transformants with TaLEA2 seemed to be more tolerant to hyperosmotic and freezing stress than transformants with TaLEA3. This implies that a close relationship exists between function and the number of repeats of the 11- mer amino acid motif in the group 3 LEA protein.     

植物抗逆蛋白（LEA3）22-氨基酸耐盐结构域在酵母细胞中的鉴定

大豆PM2蛋白属LEA3 （late embryogenesis abundant 3）蛋白。本文构建了编码PM2全长及含22-氨基酸结构域多肽（PM2、PM2A、PM2B和 PM2C）的酵母表达载体。转化酵母得到四种重组菌。SDS-PAGE电泳和ESI-MS/MS或MALDI-TOF/TOF质谱结果表明,重组菌可表达目标多肽。测定对照菌及重组菌在无胁迫、高盐（1.5 mol·L^-1 NaCl）和高渗透（2 mol·L^-1山梨糖）下的生长曲线。结果表明,在高盐胁迫下,四种重组菌胁迫后的恢复明显好于对照菌。多肽对高盐耐受能力的大小为:PM2C>PM2B≈PM2A≈PM2。证明大豆PM2蛋白的表达可提高酵母耐盐性,且22-氨基酸基序为PM2蛋白的耐盐结构域。结合前文在大肠杆菌中的结果,为“LEA蛋白可以类似机制参与原核和真核生物耐盐保护作用”的假说提供实验支持。然而,在高山梨糖胁迫下,对照菌和酵母重组菌的生长情况无明显差异。     

LEA蛋白与植物抗逆性

植物受到逆境胁迫后,LEA蛋白大量表达,可以减轻逆境引起的伤害。本文对LEA蛋白的种类、特性和功能,LEA蛋白基因结构及其表达调控,以及LEA基因表达和LEA蛋白积累与植物抗逆性的关系等方面的研究进展作了简要综述。     

脱水素在植物低温胁迫响应中的作用

脱水素(dehydrin)是一类胚胎发育后期丰富蛋白(LEA.late embryogenesis abundant proteins),含有富含赖氨酸的K片段,属于具有高度热稳定性的亲水性蛋白,在植物脱水条件下能保护细胞内蛋白质和膜结构免受破坏.低温胁迫下,耐低温植物细胞内部会发生一系列的生理生化反应来抵御低温所造成的伤害.很多研究表明植物脱水素的表达和积累与多种双子叶植物(包括草本和木本植物)以及冬季栽培的禾本科植物品种(特别是小麦和大麦)的耐低温能力密切相关.本文对近年来国内外关于脱水素的结构、功能以及内源ABA(abscisie acid)含量、拟南芥CBF(C-repeat binding factor)同源转录激活因子、春化基因、光周期信号等对脱水素基因的表达调控机制进行综述.     

胚胎发育晚期丰富蛋白(LEA蛋白)与植物抗逆性研究进展

胚胎发育晚期丰富蛋白(LEA蛋白)在自然条件下主要在种子发育晚期大量积累,植物LEA基因也在多种非生物胁迫下诱导表达。植物LEA蛋白是植物应对失水胁迫(包括干旱、盐碱、冷冻等)逆境的一种广泛存在的亲水性应答蛋白,具有很强的热稳定性。本论文就LEA蛋白的结构、分类、功能及抗逆性分子机制进行了概述与总结,为分离新的LEA蛋白成员,进行功能分析以及进一步发掘其潜在应用价值提供参考。     

异源表达CkLEA1基因增强了拟南芥对非生物胁迫的耐受性

植物在生长过程中会受到各种非生物胁迫的伤害,导致生长发育和产量受到严重影响,胚胎晚期丰富蛋白（late embryogenesis abundant proteins,LEA蛋白）在植物抵抗非生物胁迫过程中起着重要的保护作用。在前期的研究基础上,将受多种胁迫诱导的柠条锦鸡儿CkLEA1（GenBank登录号KC309408）基因转入野生型拟南芥,通过实时荧光定量PCR从7株T₃代纯合体中筛选出3个转基因株系做进一步研究。种子萌发率实验发现,在200 mmol/L NaCl和400 mmol/L甘露醇处理下,转基因株系萌发率均高于野生型拟南芥。干旱处理2周大的幼苗后,转基因株系明显比野生型更抗旱,存活率高于野生型,并且失水率低于野生型。同时,转基因株系积累了较少的丙二醛（MDA）,超氧化物歧化酶（SOD）活性和谷胱甘肽（GSH）含量也高于野生型。这些结果表明,柠条锦鸡儿CkLEA1基因在种子萌发阶段提高了拟南芥对盐和渗透胁迫的耐受性,并且提高了转基因拟南芥幼苗生长阶段对干旱胁迫的抵抗能力。     

Soybean PM2 Protein (LEA3) Confers the Tolerance of Escherichia coli and Stabilization of Enzyme Activity Under Diverse Stresses

Late embryogenesis abundant (LEA) proteins are closely associated with the tolerance of diverse stresses in organisms. To elucidate the function of group 3 LEA proteins, the soybean PM2 protein (LEA3) was expressed in E. coli and the protective function of the PM2 protein was assayed both in vivo and in vitro. The results of a spot assay and survival ratio demonstrated that the expression of the PM2 protein conferred the tolerance to the E. coli recombinant for different temperature conditions (4, −20 or 50°C) or high-salinity stresses (120 mmol/l MgCl₂ or 120 mmol/l CaCl₂). In addition, it was demonstrated that the in vitro addition of the PM2 protein could prevent the lactate dehydrogenase (LDH) inactivation normally induced by freeze–thaw. In the 62°C condition, the PM2 protein (1:5 mass ratio to LDH) effectively prevented the LDH thermo-denaturation by acting synergistically with trehalose (62.5 μg/ml), although the PM2 protein alone at this concentration showed little protective effect on LDH activity. Furthermore, the results showed that the PM2 protein could partially prevent the thermo-denaturation of the bacterial proteome after boiling for 2 min. Based on these results, we propose that the PM2 protein itself, or together with trehalose, conferred the tolerance to the E. coli recombinant against diverse stresses by protecting proteins and enzyme activity under low- or high- temperature conditions.     

长春花Crlea基因的克隆及原核表达初步分析

晚期胚胎丰富（Late Embryogenesis Abundant, LEA）蛋白是植物在干旱胁迫下响应并被描述为具有潜在的抗旱功能的一类重要的抗旱蛋白。通过建立干旱胁迫下长春花（Catharanthus roseus）的cDNA文库并进行测序筛选分析,首次分离得到Crlea（Crlea for Catharanthus roseus late embryogenesis abundant）全长基因。该基因具有492 bp的开放读码框,编码163个氨基酸,其中偏性氨基酸含量占总蛋白的55.9%。同源性分析表明该假定蛋白与胡萝卜（Daucus carota）LEA DC3 的同源性达69%。亲水性分析表明具有极强的亲水性。为进一步验证CrLEA蛋白的功能,构建了Crlea基因的原核表达载体并在大肠杆菌中对其表达进行了分析。结果表明,原核载体成功的表达了CrLEA蛋白,亲水性实验及热稳定性实验表明CrLEA蛋白具有极强的亲水性和热稳定性。     

Cryoprotective activities of group 3 late embryogenesis abundant proteins from Chlorella vulgaris C-27

The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC61) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1lambdaT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins in E. coli, then purified. The two HIC proteins were found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1 x 10(6) times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.     

第三组胚胎晚期丰富蛋白lea3基因研究概述

晚期胚胎富集蛋白(late embryogenesis abundant protein,LEA蛋白)是在高等植物胚胎发育晚期大量积累的一类蛋白,根据其结构特点LEA蛋白一般分为6组,其中第3组LEA蛋白(LEA3)含有11个氨基酸串联重复的基元序列,可以形成α-螺旋结构,能在干旱胁迫的环境中保护生物大分子,减轻水份胁迫对植物造成的伤害,与植物抗逆性密切相关。该文就lea3基因及其蛋白的结构、功能、基因表达和应用等进行简要的综述,并对lea3基因及其蛋白今后的研究方向和应用前景进行了展望。     

菘蓝IiLEA基因的克隆及胁迫表达分析

胚胎发育晚期丰富蛋白(LEA蛋白),是指胚胎发育后期种子中大量积累的一系列蛋白质,大量研究表明这些蛋白质的积累与渗透胁迫耐受性密切相关.该实验以250 mmol/L NaCl溶液处理9 h的菘蓝幼苗为材料,经RT-PCR方法扩增得到LEA基因,命名为IiLEA.经测序确认该基因含有648 bp的开放阅读框,编码由215个氨基酸组成的亲水性蛋白质.对培养30 d的菘蓝无菌幼苗进行自然干旱处理和盐胁迫处理,Northern杂交结果显示,正常生长条件下,IiLEA基因在菘蓝幼苗体内不表达,随着胁迫时间的延长其表达量逐渐增加,到9 h时表达量达到峰值,干旱处理和盐处理有相似结果,表明该基因可能受逆境胁迫诱导表达.     

Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit

The late embryogenesis abundant (LEA) proteins are plant proteins that are synthesized at the onset of desiccation in maturing seeds and in vegetative organs exposed to water deficit. Here, we show that most LEA proteins are comprised in a more widespread group, which we call "hydrophilins." The defining characteristics of hydrophilins are high glycine content (>6%) and a high hydrophilicity index (>1.0). By data base searching, we show that this criterion selectively differentiates most known LEA proteins as well as additional proteins from different taxons. We found that within the genomes of Escherichia coli and Saccharomyces cerevisiae, only 5 and 12 proteins, respectively, meet our criterion. Despite their deceivingly loose definition, hydrophilins usually represent <0.2% of the proteins of a genome. Additionally, we demonstrate that the criterion that defines hydrophilins seems to be an excellent predictor of responsiveness to hyperosmosis since most of the genes encoding these proteins in E. coli and S. cerevisiae are induced by osmotic stress. Evidence for the participation of one of the E. coli hydrophilins in the adaptive response to hyperosmotic conditions is presented. Apparently, hydrophilins represent analogous adaptations to a common problem in such diverse taxons as prokaryotes and eukaryotes.     

Insights into Maize LEA proteins: from proteomics to functional approaches

LEA (late embryogenesis abundant) proteins participate in plant stress tolerance responses, but the mechanisms by which protection occurs are not fully understood. In the present work the unfolded proteins from maize dry embryos were analyzed by mass spectrometry. Twenty embryo proteins were identified, and among them 13 corresponded to LEA-type proteins. We selected three major LEA proteins, Emb564, Rab17 and Mlg3, belonging to groups 1, 2 and 3, respectively, and we undertook a comparative study in order to highlight differences among them. The post-translational modifications of native proteins were analyzed and the anti-aggregation properties of recombinant Emb564, Rab17 and Mgl3 proteins were evaluated in vitro. In addition, the protective effects of the LEA proteins were assessed in living cells under stress in Escherichia coli cells and in Nicotiana bentamiana leaves agroinfiltrated with fluorescent LEA-green fluorescent protein (GFP) fusions. Protein visualization by confocal microscopy indicated that cells expressing Mg3-GFP showed reduced cell shrinkage effects during dehydration and that Rab17-GFP co-localized to leaf oil bodies after heat shock. Overall, the results highlight differences and suggest functional diversity among maize LEA groups.     

Diverse LEA (late embryogenesis abundant) and LEA-like genes and their responses to hypersaline stress in post-diapause embryonic development of Artemia franciscana

From post-diapause cysts of Artemia franciscana, we defined fourteen LEA (late embryogenesis abundant) and LEA-like genes, including four novel members (Afrlea1-5, Afrlea3-5, Afrlea3-like1 and Afrlea3-like2), which were classified into four groups: G1, G3, G3-like (LEA group3-like), and SMP-like (seed-maturation-protein-like), based on their conserved and diversified sequence motifs and amino acid compositions among bacteria, plants, and animals. We also validated six representative genes based on quantitative RT-PCR, including three LEA and two LEA3-like genes that are down-regulated when dehydrated cysts hatch to desiccation-intolerant larvae as well as one SMP-like gene that is slightly up-regulated. We further tested their responses to hypersaline stress for four representatives-one from each group-and found that the expression of Afrlea1-5 and Afrlea3-2 were inducible but not Afrlea3-like1 and Afrsmp-like. This result suggested that the LEA and LEA-like genes may play different roles in resistance to hypersaline stress.     

脱水素研究进展

脱水素(dehydrin)是植物体内的一种LEA蛋白,能够在植物胚胎发育后期以及逆境下大量表达,广泛存在于植物界。它是具有高度热稳定性的亲水性蛋白,有三类非常保守的区域,即K,Y和S片段。依据这三类片段的组成情况,可将脱水素分为5个基本类别。脱水素可通过多种转运方式定位于植物细胞的不同部位,以行使其功能。其基因的表达存在依赖ABA和不依赖ABA两种途径,并且受到多种环境因素的影响,能稳定细胞膜和许多大分子的结构以避免脱水对细胞造成的伤害。近年来,脱水素的结构和组成、在细胞中的定位及转运、基因的表达与调控、功能与作用机理等方面的研究已取得了很大的进展。     

大麦Dhn6基因的克隆、蛋白质结构预测与干旱胁迫表达模式

脱水素(Dehydrins,DHNs)是高等植物胚胎发育晚期产生的一类特异多肽,其表达累积程度与植物的发育阶段、低温、ABA和脱水信号调节等因素密切相关。为了解脱水素的结构与干旱胁迫表达累积反应,文章从六棱大麦分离到序列全长为1 767 bp的Dhn6基因,序列分析结果表明,该基因含一个92 bp内含子,90~1 759 bp为一个开放阅读框,与裸大麦Dhn6基因(GenBank登录号:AF043091)的同源性最高,达93.18%,编码523个氨基酸残基的多肽,预测蛋白质的分子量为49.68 kDa,理论等电点为8.04。结构分析发现,蛋白质具有3个螺旋区,无规则卷曲构成二级结构的主要组分,亲水氨基酸比例超过83%;三维结构预测发现,多肽链自身反向平行排列成松散的亲水索链,K-片段参与兼性?-螺旋结构域的形成,意味着该脱水素具有束缚自由水、稳定细胞膜相结构的功能。实时定量RT-PCR检测结果表明,Dhn6基因的相对表达水平在干旱处理8 h快速累积,推测DHN6在大麦对干旱胁迫的早期响应中发挥重要功能。