转柽柳晚期胚胎富集蛋白基因烟草T1代的耐盐性评价

目的:验证转柽柳晚期胚胎富集(LEA)蛋白基因烟草T1代的耐盐性。方法:采用盐胁迫方式,对转柽柳LEA蛋白基因烟草T1代的6个株系及非转基因对照烟草T1代进行不同浓度NaCl胁迫处理,分析了NaCl胁迫下转基因烟草的生长量、根系的发育及盐害程度。结果:各转基因烟草T1代组培苗在150mmol/L的NaCl培养基上根系生长良好,平均增重(鲜重)是非转基因对照的7.72倍,平均高生长是非转基因对照的3.51倍,盐害指数低于或等于50%;而非转基因对照烟草T1代组培苗生长缓慢,根系几乎不能生长发育,盐害指数达65%。结论:柽柳LEA蛋白基因的导入提高了T1代烟草的耐盐性。     

DELLA蛋白缺失对拟南芥干旱胁迫耐受性的影响

为探索DELLA蛋白缺失对拟南芥耐旱能力的影响,对拟南芥野生型Ler和DELLA蛋白缺失突变体della进行干旱处理,测定存活率、萌发率、离体叶片的失水率、脯氨酸、可溶性糖和丙二醛含量,并对发挥植物细胞脱水保护功能的胚胎晚期丰富蛋白编码基因LEA和ABA应答基因LOX3、COR15b、COR413的表达量进行了检测。结果表明:(1)干旱21d后复水,della突变体的存活率明显高于野生型Ler;(2)della突变体在含甘露醇的固体培养基上的萌发率显著高于Ler;(3)della突变体离体叶片的失水速率明显低于Ler;(4)干旱胁迫后,della突变体脯氨酸、可溶性糖和丙二醛含量的积累低于Ler;(5)干旱胁迫后,della突变体的LEA基因上调表达程度高于Ler,而ABA应答基因上调表达程度低于Ler。研究表明,DELLA蛋白的缺失有助于提高植物抗旱能力。     

荧光定量PCR检测干旱胁迫下长春花Crlea基因的相对表达

首次从长春花中克隆了Crlea（Crlea for Catharanthus roseus late embryogenesis abundant）的全长基因,采用荧光定量PCR方法对干旱胁迫下长春花叶片和根部Crlea基因的表达模式进行监测,结果表明,在0.5～8 h的胁迫时间中,叶片和根部的Crlea基因表现出相似的积累模式。长春花Crlea基因的表达随着胁迫时间的延长而表达增强。在叶片中,在6 h和8 h的干旱处理后,Crlea基因表达显著提高,分别是未处理材料的9.984和20.431倍。在根部, 在8 h的处理后,Crlea基因的表达量显著提高（2.831倍于对照)。初步结果表明Crlea基因的表达没有组织特异性,并且为干旱胁迫正调控。     

杜仲叶片干旱胁迫响应相关差异蛋白的筛选与鉴定

为研究干旱胁迫下杜仲幼苗生理生化及分子响应机制,利用盆栽试验,通过持续（3、6、9、12、15 d）干旱胁迫处理和复水处理,研究杜仲幼苗的生理响应特性。同时,通过研究对照与处理15 d后的杜仲幼苗差异蛋白质组,分析杜仲幼苗对干旱胁迫的分子响应机制。结果表明,随着干旱处理时间的延长,杜仲叶片的水分饱和亏逐渐增加;光合速率、蒸腾速率、胞间二氧化碳浓度、气孔导度均逐渐减小;SOD、POD、CAT活性呈先上升后降低的趋势;丙二醛含量则呈现先上升,然后下降,最后又上升的变化特点;脯氨酸和可溶性糖含量的变化趋势与SOD等活性变化一致,前期上升,后期下降。在复水后,杜仲叶片的所有指标均有所恢复,但未达到干旱处理之前的水平。表明干旱胁迫影响了杜仲叶片的正常生长代谢。通过对干旱处理15 d后杜仲叶片总蛋白进行双向电泳分离和MALDI-TOF-TOF生物质谱鉴定,成功鉴定出36个差异表达蛋白,其中22个上调表达,14个下调表达。对36个差异蛋白进行功能分析发现,这些差异蛋白主要涉及信号传导、光合作用、碳代谢、能量代谢、次级代谢物合成、抗氧化保护酶、氨基酸代谢和蛋白质代谢。推测杜仲为适应干旱胁迫,首先是感应干旱胁迫信号,并传导至细胞内,影响杜仲叶片中光合作用、次级代谢物合成和蛋白质的生物合成;同时,通过过氧化物保护酶的作用,将过多活性氧加以清除;另一方面,则是通过增强糖酵解,磷酸戊糖途径,产生能量供杜仲正常生长所需。从生理机制来看,杜仲叶片同过增加胞内脯氨酸、可溶性糖含量,降低胞内渗透势,减少叶片中水分损失,与氨基酸合成和糖代谢相关蛋白的表达量上升的结果一致。     

干旱和盐胁迫对马铃薯试管苗亚细胞结构及生理生化指标的影响

以‘陇薯3号’脱毒试管苗为材料,研究了不同浓度PEG-4000(0、2%、4%、6%、8%)和NaCl(0、25、50、100、200mmol/L)对马铃薯2周大小试管苗根系生长、叶肉细胞超微结构及部分生理生化指标的影响,为筛选耐盐抗旱马铃薯种质提供理论依据。结果显示:(1)随着PEG-4000和NaCl浓度的增加,马铃薯试管苗根总长、根体积、根数均呈现下降趋势,并且胁迫浓度越高时间越长其下降趋势越明显,而盐胁迫处理的下降幅度明显大于PEG胁迫处理。(2)随着PEG-4000和NaCl浓度的增加,马铃薯试管苗叶肉细胞细胞壁明显变厚,发生明显的质壁分离,嗜锇颗粒显著增加,出现大量囊泡,叶绿体损害逐渐加剧,直至完全解体。(3)随着PEG-4000和NaCl浓度的增加,马铃薯试管苗脯氨酸(Pro)含量显著增加,过氧化氢(CAT)和超氧化物歧化酶(SOD)活性显著增强,而其丙二醛(MDA)含量迅速增加,但叶片叶绿素含量持续下降。研究表明,在PEG-4000模拟干旱和NaCl胁迫条件下,马铃薯试管苗叶片叶绿体结构受到严重损害,叶绿素含量显著降低,且胁迫程度越强损害越严重、下降幅度越大;同时,干旱和高盐胁迫也诱导马铃薯试管苗脯氨酸含量和抗氧化酶CAT和SOD活性显著上调,一定程度上缓解了干旱和高盐胁迫的伤害。     

干旱胁迫与小麦幼苗亚精胺代谢变化的关系

通过对亚精胺（spermidine,Spd）在抗旱性不同的小麦品种幼苗中的作用的研究,发现抗旱品种周麦18号在渗透胁迫处理时,其叶片中的Spd含量明显大于不抗旱的豫麦51号。用Spd合成的抑制剂甲基乙二醛-双（鸟嘌呤腙MGBG）处理周麦18号,则导致Spd含量下降和抗性的降低,外源Spd又可逆转MGBG对周麦18号在渗透胁迫下的伤害。外源Spd可以明显提高豫麦51号的叶片内Spd含量,并相应提高其抗性。以上结果表明,Spd可以提高小麦幼苗的抗渗透胁迫能力。     

固有无序蛋白研究进展及其对细胞胁迫耐受性的影响

固有无序蛋白(intrinsically disordered proteins,IDPs)是指在生理条件下缺乏有序稳定的高级结构,整体或局部不折叠,但能够参与多种生物学过程、行使特定生物学功能的一类蛋白质.固有无序蛋白决定了其不同于经典蛋白质"序列-结构-功能"的功能范式,丰富了蛋白质"结构-功能"的多样性.固有无序...     

栓皮栎幼苗对土壤干旱胁迫的生理响应

以栓皮栎一年生盆栽苗为实验材料,采用称重控水的方法,设置不同土壤水分胁迫梯度,系统分析其幼苗在不同干旱胁迫条件下的生理生化响应特征,以探索栓皮栎耐旱特性.结果显示:(1)栓皮栎幼苗叶片中3种保护酶(SOD、POD、CAT)活性在对照(CK,土壤相对含水量19.5％～21.5％)条件下保持稳定,而中度干旱(T2,9.5％～11.5％)和重度干旱(T3,5.5％～7.5％)条件下,随着胁迫时间的延长呈先增高后降低的趋势,且变化的幅度在不同胁迫强度下存在差异.(2)在整个干旱胁迫过程中,各胁迫处理叶片丙二醛(MDA)含量均呈上升趋势,不同胁迫强度的变化幅度不同;叶片中的可溶性蛋白含量和根系活力随着干旱胁迫程度的增强呈先增高后降低的趋势.(3)栓皮栎幼苗叶片的脯氨酸含量随着干旱胁迫时间的延长表现出先增加后降低的趋势;叶片叶绿素a、叶绿素b、总叶绿素含量以及叶绿素a/b值均呈逐渐降低的趋势.研究表明,栓皮栎幼苗在短期和轻度干旱胁迫下通过提高自身的保护酶活性、增加可溶性蛋白和脯氨酸含量、提高根系活力等来抵御干旱环境的伤害,从而表现出较强的耐旱特性;而在重度干旱胁迫条件下,栓皮栎幼苗自我调节能力丧失,体内代谢紊乱,导致保护酶活性、可溶性蛋白、脯氨酸含量和根系活力等下降,从而受到干旱伤害.     

干旱胁迫与ABA的信号转导

植物经历干旱胁迫时,ABA被普遍认为是一种干旱信号而传递干旱信息.在干旱信号ABA的转导过程中,从ABA的被感知到保卫细胞发生变化引起气孔关闭以及ABA诱导的基因表达都经历了复杂的变化.本文对ABA的信号转导过程进行了综述.     

丹参晚期胚胎蛋白基因SmLEA的克隆及表达分析

对丹参EST序列进行Blast分析,发现一条序列与晚期胚胎丰富蛋白(late embryogenesis abundant)基因有较高的相似性,在此基础上设计引物,分别从cDNA和gDNA水平克隆到该基因的全长(Genbank注册号:AY725206),命名为SmLEA.该序列全长739 bp,无内含子,包含1个长为495 bp的开放阅读框,编码164个氨基酸.序列比对结果显示,该序列与番茄的晚期胚胎丰富蛋白Lemmi9有较高的相似性(69%),推测该编码蛋白属于晚期胚胎蛋白LEA14家族成员.生物信息学显示,SmLEA所编码蛋白SmLEA的相对分子质量为17.34 kD,理论等电点为4.51,富含天冬氨酸及AS、IP、KV、VS、TIP肽段,定位于细胞质中,为稳定类蛋白.实时荧光定量PCR结果显示,该基因在丹参的根、茎、叶中均有表达,为组成型表达基因.     

Dehydration-regulated processing of late embryogenesis abundant protein in a desiccation-tolerant nematode

Late embryogenesis abundant (LEA) proteins occur in desiccation-tolerant organisms, including the nematode Aphelenchus avenae, and are thought to protect other proteins from aggregation. Surprisingly, expression of the LEA protein AavLEA1 in A. avenae is partially discordant with that of its gene: protein is present in hydrated animals despite low cognate mRNA levels. Moreover, on desiccation, when its gene is upregulated, AavLEA1 is specifically cleaved to discrete, smaller polypeptides. A processing activity was found in protein extracts of dehydrated, but not hydrated, nematodes, and main cleavage sites were mapped to 11-mer repeated motifs in the AavLEA1 sequence. Processed polypeptides retain function as protein anti-aggregants and we hypothesise that the expression pattern and cleavage of LEA protein allow rapid, maximal availability of active molecules to the dehydrating animal.     

The in vitro structure and functions of the disordered late embryogenesis abundant three proteins

Late embryogenesis abundant (LEA) proteins are produced during seed embryogenesis and in vegetative tissue in response to various abiotic stressors. A correlation has been established between LEA expression and stress tolerance, yet their precise biochemical mechanism remains elusive. LEA proteins are very rich in hydrophilic amino acids, and they have been found to be intrinsically disordered proteins (IDPs) in vitro. Here, we perform biochemical and structural analyses of the four LEA3 proteins from Arabidopsis thaliana (AtLEA3). We show that the LEA3 proteins are disordered in solution but have regions with propensity for order. All LEA3 proteins were effective cryoprotectants of LDH in the freeze/thaw assays, while only one member, AtLEA3‐4, was shown to bind Cu²⁺ and Fe³⁺ ions with micromolar affinity. As well, only AtLEA3‐4 showed binding and a gain in α‐helicity in the presence of the membrane mimic dodecylphosphocholine (DPC). We explored this interaction in greater detail using ¹⁵N‐heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance, and demonstrate that two sets of conserved motifs present in AtLEA3‐4 are involved in the interaction with the DPC micelles, which themselves gain α‐helical structure.     

The major‐effect quantitative trait locus Fnl7.1 encodes a late embryogenesis abundant protein associated with fruit neck length in cucumber

Fruit neck length (FNL) is an important quality trait in cucumber because it directly affects its market value. However, its genetic basis remains largely unknown. We identified a candidate gene for FNL in cucumber using a next‐generation sequencing‐based bulked segregant analysis in F₂ populations, derived from a cross between Jin5‐508 (long necked) and YN (short necked). A quantitative trait locus (QTL) on chromosome 7, Fnl7.1, was identified through a genome‐wide comparison of single nucleotide polymorphisms between long and short FNL F₂ pools, and it was confirmed by traditional QTL mapping in multiple environments. Fine genetic mapping, sequences alignment and gene expression analysis revealed that CsFnl7.1 was the most likely candidate Fnl7.1 locus, which encodes a late embryogenesis abundant protein. The increased expression of CsFnl7.1 in long‐necked Jin5‐508 may be attributed to mutations in the promoter region upstream of the gene body. The function of CsFnl7.1 in FNL control was confirmed by its overexpression in transgenic cucumbers. CsFnl7.1 regulates fruit neck development by modulating cell expansion. Probably, this is achieved through the direct protein–protein interactions between CsFnl7.1 and a dynamin‐related protein CsDRP6 and a germin‐like protein CsGLP1. Geographical distribution differences of the FNL phenotype were found among the different cucumber types. The East Asian and Eurasian cucumber accessions were highly enriched with the long‐necked and short‐necked phenotypes, respectively. A further phylogenetic analysis revealed that the Fnl7.1 locus might have originated from India. Thus, these data support that the CsFnl7.1 has an important role in increasing cucumber FNL.     

A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state

Late embryogenesis abundant (LEA) proteins are a highly diverse group of polypeptides expected to play important roles in desiccation tolerance of plant seeds. They are also found in other plant tissues and in some anhydrobotic invertebrates, fungi, protists and prokaryotes. The LEA protein LEAM accumulates in the matrix space of pea (Pisum sativum) mitochondria during late seed maturation. LEAM is an intrinsically disordered protein folding into amphipathic α-helix upon desiccation. This suggests that it could interact with the inner mitochondrial membrane, providing structural protection in dry seeds. Here, we have used Fourier-transform infrared and fluorescence spectroscopy to gain insight into the molecular details of interactions of LEAM with phospholipid bilayers in the dry state and their effects on liposome stability. LEAM interacted specifically with negatively charged phosphate groups in dry phospholipids, increasing fatty acyl chain mobility. This led to an enhanced stability of liposomes during drying and rehydration, but also upon freezing. Protection depended on phospholipid composition and was strongly enhanced in membranes containing the mitochondrial phospholipid cardiolipin. Collectively, the results provide strong evidence for a function of LEAM as a mitochondrial membrane protectant during desiccation and highlight the role of lipid composition in the interactions between LEA proteins and membranes.     

A Putative LEA Protein,but no Trehalose,is Present in Anhydrobiotic Bdelloid Rotifers

Some eukaryotes, including bdelloid rotifer species, are able to withstand desiccation by entering a state of suspended animation. In this ametabolic condition, known as anhydrobiosis, they can remain viable for extended periods, perhaps decades, but resume normal activities on rehydration. Anhydrobiosis is thought to require accumulation of the non-reducing disaccharides trehalose (in animals and fungi) or sucrose (in plant seeds and resurrection plants), which may protect proteins and membranes by acting as water replacement molecules and vitrifying agents. However, in clone cultures of bdelloid rotifers Philodina roseola and Adineta vaga, we were unable to detect trehalose or other disaccharides in either control or dehydrating animals, as determined by gas chromatography. Indeed, trehalose synthase genes (tps) were not detected in these rotifer genomes, suggesting that bdelloids might not have the capacity to produce trehalose under any circumstances. This is in sharp contrast to other anhydrobiotic animals such as nematodes and brine shrimp cysts, where trehalose is present during desiccation. Instead, we suggest that adaptations involving proteins might be more important than those involving small biochemicals in rotifer anhydrobiosis: on dehydration, P. roseola upregulates a hydrophilic protein related to the late embryogenesis abundant (LEA) proteins associated with desiccation tolerance in plants. Since LEA-like proteins have also been implicated in the desiccation tolerance of nematodes and micro-organisms, it seems that hydrophilic protein biosynthesis represents a common element of anhydrobiosis across several biological kingdoms.     

<Emphasis Type="Italic">OsLEA3</Emphasis>, a late embryogenesis abundant protein gene from rice,confers tolerance to water deficit and salt stress to transgenic rice

OsLEA3 is a late embryogenesis abundant group 3 protein. The OsLEA3 gene located on chromosome 5 of rice (Oryza sativa L.) includes one intron and two exons and encodes a protein of 200 amino acid residues. Expression analysis revealed that OsLEA3 was induced by water deficit and salt stress. Overexpression of the OsLEA3 gene in the transgenic rice plants allowed us to test the role of the OsLEA3 protein in stress tolerance. The accumulation of the OsLEA3 protein in the vegetative tissues of transgenic rice plants enhanced their tolerance to water deficit and salt stress. These results demonstrate a role for the OsLEA3 protein in stress protection and suggest the potential of the OsLEA3 gene for genetic engineering of stress tolerance.     

Temporal profiling of the heat-stable proteome during late maturation of Medicago truncatula seeds identifies a restricted subset of late embryogenesis abundant proteins associated with longevity

Developing seeds accumulate late embryogenesis abundant (LEA) proteins, a family of intrinsically disordered and hydrophilic proteins that confer cellular protection upon stress. Many different LEA proteins exist in seeds, but their relative contribution to seed desiccation tolerance or longevity (duration of survival) is not yet investigated. To address this, a reference map of LEA proteins was established by proteomics on a hydrophilic protein fraction from mature Medicago truncatula seeds and identified 35 polypeptides encoded by 16 LEA genes. Spatial and temporal expression profiles of the LEA polypeptides were obtained during the long maturation phase during which desiccation tolerance and longevity are sequentially acquired until pod abscission and final maturation drying occurs. Five LEA polypeptides, representing 6% of the total LEA intensity, accumulated upon acquisition of desiccation tolerance. The gradual 30-fold increase in longevity correlated with the accumulation of four LEA polypeptides, representing 35% of LEA in mature seeds, and with two chaperone-related polypeptides. The majority of LEA polypeptides increased around pod abscission during final maturation drying. The differential accumulation profiles of the LEA polypeptides suggest different roles in seed physiology, with a small subset of LEA and other proteins with chaperone-like functions correlating with desiccation tolerance and longevity.