小桐子JcLEA4基因克隆及表达和功能分析

LEA蛋白是一类跟抗逆性紧密相关的蛋白,在不同物种中都有大量发现。基于前期对小桐子叶片在干旱锻炼和干旱胁迫下的RNA-seq分析,本研究分离出一个LEA编码基因(XM_012232372.1),经序列分析可知该蛋白基因CDS序列全长为459 bp,编码蛋白由152个氨基酸组成。多重序列比对和进化分析显示该蛋白属于第四组典型亲水性LEA蛋白,暂命名为Jc LEA4。荧光定量PCR分析发现,幼苗叶片中Jc LEA4转录本的积累受到空气干旱胁迫和外源ABA处理的显著诱导。在酿酒酵母中异源表达分析显示,Jc LEA4的过表达提高了转基因酵母对PEG模拟干旱胁迫的存活率和生长速率,表现出增强的耐受性。本研究初步证实了Jc LEA4基因与抗旱性紧密相关,为以后小桐子的抗旱性遗传改良提供了良好的基因资源。     

LEA蛋白研究进展

LEA蛋白(late embriogenesis abundant protein,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、ABA、紫外辐射和NaHCO3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.     

柠条锦鸡儿CkLEA1基因克隆及表达分析

植物受到逆境胁迫后,大量逆境响应基因会被诱导表达,LEA蛋白编码基因就是与植物抗旱、抗冷等非生物胁迫密切相关的一类基因.从已构建的柠条锦鸡儿干旱胁迫抑制性削减杂交文库中筛选到了一条LEA蛋白编码基因并进行了克隆.序列比对与系统进化分析显示该基因属于LEA3基因家族成员,命名为CkLEA1(GenBank登录号是KC309408).克隆得到该基因gDNA长469bp,包含两个外显子和一个内含子;cDNA长357bp,包含300bp的开放阅读框,推导编码99个氨基酸的蛋白质.利用荧光定量PCR技术对CkLEA1基因在各种逆境胁迫条件的表达情况进行初步研究表明,CkLEA1受干旱、ABA、冷、热、盐和碱等处理不同程度地诱导,推测其与柠条锦鸡儿响应逆境胁迫的机制有关.     

香蕉钙调蛋白基因MaCAM在非生物胁迫下的表达分析

为研究香蕉钙调蛋白基因MaCAM的功能,对香蕉幼苗进行盐、低温和干旱胁迫处理,利用荧光定量PCR技术分析香蕉根中钙调蛋白基因MaCAM在上述不同非生物胁迫条件下的表达特性.盐胁迫处理后MaCAM表达随时间延长为先增加后降低趋势,在处理4 h时表达量最高;低温胁迫处理后,MaCAM的表达量随温度降低呈梯度增加,在温度降至5℃时表达量在处理范围内达最大值;干旱胁迫处理后,MaCAM的表达量随胁迫程度增加也是呈梯度增加,重度干旱胁迫时在处理范围内表达量最高.说明香蕉中的钙调蛋白基因具有响应非生物胁迫的能力,可能在香蕉适应盐、低温和干旱等胁迫逆境中发挥重要作用,参与了香蕉幼苗抗逆性调控.     

日本结缕草耐逆基因ZjLEA2的克隆及功能探究

植物胚胎发育晚期丰富蛋白(Late embryogenesis abundant)在抵御缺水胁迫中起重要作用.本研究从日本结缕草'Meyer'品种中克隆获得了 1个LEA蛋白基因,命名为ZjLEA2.该基因编码区长501个碱基,推测编码的蛋白质含166个氨基酸,预测分子量17kD,等电点9.10,具有LEA蛋白家族第二组的典型结构特征,亲水性强.基因的系统发育树分析结果显示,ZjLEA2蛋白与耐旱的禾本科草种狗牙根和无芒隐子草的同源基因遗传距离最近.ZjLEA2基因的表达受干旱、低温和高盐胁迫诱导,表达量峰值均出现在胁迫72 h后.转化ZjLEA2基因的酵母细胞对冷冻低温和高盐胁迫的耐受能力明显增强.本研究结果说明ZjLEA2基因具有抗逆功能,在结缕草抗非生物胁迫中后期起作用.     

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。     

天山雪莲LEA14基因克隆及功能分析

从天山雪莲叶片低温诱导的EST文库中获得了1个胚胎发育晚期丰富蛋白基因(LEA)cDNA全长序列。序列分析表明,该基因含有1个468bp编码155个氨基酸的开放阅读框。NCBI保守域预测此蛋白属于LEA_2家族,命名为SiLEA14。系统进化分析表明,该蛋白与北柴胡的LEA-2蛋白亲缘关系最近。荧光定量PCR结果显示,SiLEA14表达量在低温、盐和干旱胁迫条件下迅速升高。亚细胞定位结果表明,SiLEA14蛋白定位于细胞核中。利用农杆菌介导法将该基因导入烟草,测定并分析转基因植株在冷冻和盐胁迫处理下的生理指标,结果表明,SiLEA14基因在烟草中的过量表达提高了烟草的抗冻和耐盐能力。     

小麦TaLEA1基因的表达特征及抗逆功能验证

我国土壤盐碱化日益严重,对我国的粮食安全造成了严重威胁,因此耐盐基因挖掘对作物耐盐育种非常重要。许多研究表明胚胎发育晚期丰富蛋白(LEA)在植物应对非生物胁迫中发挥积极作用。本研究以小麦TaLEA1基因为研究对象,分析了其表达蛋白的理化性质及基因表达模式,并通过在拟南芥中过表达,分析Ta LEA1基因的抗逆功能。结果表明,TaLEA1基因的表达蛋白属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。Ta LEA1基因在小麦根、茎、叶、花、种子等不同组织中均有表达,盐胁迫条件诱导其高表达。在拟南芥中过表达TaLEA1基因,显著提高了盐胁迫下转基因拟南芥的种子萌发率、根长及盐和旱胁迫下的叶绿素含量。本研究结果为LEA基因抗逆机理的研究和耐盐基因的挖掘提供了重要信息。     

硅对辣椒CaLEA5基因低温胁迫下表达分析

通常在逆境胁迫下,植物细胞会诱导产生一系列的逆境蛋白来保护细胞免受伤害,其中胚胎发育晚期丰富(LEA)蛋白是目前研究最为普遍的一种。为了研究辣椒LEA蛋白的耐寒性,以豫椒101为材料,根据已经报道的LEA基因序列设计引物,通过PCR技术从辣椒基因组中获得LEA基因,命名为CaLEA5。经生物信息学分析,该基因具有一个完整的ORF为297 bp,编码98个氨基酸。辣椒CaLEA5蛋白质含有LEA基因家族保守的结构域。对其亚细胞定位、跨膜结构进行分析,预测其定位在叶绿体,存在跨膜结构。荧光定量PCR检测结果表明,低温胁迫可诱导CaLEA5基因的表达,且其表达量在迅速达到峰值后又降低,说明CaLEA5是一个快速响应基因,推测其在辣椒抗逆机制中起着重要的作用。在低温胁迫的条件下,硅处理可延缓CaLEA5基因的上调表达。     

植物抗旱和耐重金属基因工程研究进展

干旱和重金属污染严重影响植物的生长发育.植物耐逆相关基因的克隆和功能鉴定研究,为通过基因工程途径提高植物的抗逆性奠定了理论基础.水分亏缺、高盐、低温和重金属胁迫都能诱导LEA(late embryogenesis abundant protein)基因的表达.转基因研究表明,LEA蛋白具有抗旱保护作用、离子结合特性以及抗氧化活性;水孔蛋白存在于细胞膜和液泡膜上,在细胞乃至整个植物体水分吸收和运输过程中发挥重要作用.干旱和盐胁迫促进水孔蛋白基因转录物的积累.过量表达水孔蛋白可增强水分吸收和运输,提高植物的抗旱能力.金属转运蛋白参与重金属离子的吸收、运输和累积等过程.这些蛋白基因在改良草坪草植物的抗旱节水和耐重金属能力等方面具有潜在的应用价值.     

Accumulation of LEA proteins in salt (NaCl) stressed young seedlings of rice (Oryza sativa L.) cultivar Bura Rata and their degradation during recovery from salinity stress

Germination and subsequent hydroponic growth under salt stress (100 mmol/L NaCl) triggered an accumulation of six major stress proteins and resulted in a growth arrest of young seedlings of rice (Oryza sativa L.) cv. Bura Rata. Based on two-dimensional electrophoretic resolution, partial amino acid sequencing and immunodetection techniques, four of the salt stress-induced polypeptides were identified as LEA proteins. Under all experimental conditions wherein seedlings exhibited superior halotolerance, salt stress-induced LEA proteins were expressed at low levels. In contrast, accumulation of LEA proteins was found associated with growth arrest. When returned to non-saline media, seedlings stressed with salt for four days recovered immediately. Longer exposure to 100 mmol/L NaCl, however, progressively delayed recovery and reduced the number of seedlings which could recover from salt stress. Recovery from salt stress was consistently accompanied by degradation of the salt stress-induced LEA proteins. The results of this study show that LEA proteins accumulate during the salinity-triggered growth arrest of young Bura Rata seedlings and are mobilised during the recovery of seedlings from salinity stress.     

Photosynthetic Physiological Response of Radix Isatidis (Isatis indigotica Fort.) Seedlings to Nicosulfuron

Radix Isatidis (Isatis indigotica Fort.) is one of the most important traditional Chinese medicine plants. However, there is no suitable herbicide used for weed control in Radix Isatidis field during postemergence stage. To explore the safety of sulfonylurea herbicide nicosulfuron on Radix Isatidis (Isatis indigotica Fort.) seedlings and the photosynthetic physiological response of the plant to the herbicide, biological mass, leaf area, photosynthetic pigment content, photosynthetic rate, chlorophyll fluorescence characteristics, and P₇₀₀ parameters of Radix Isatidis seedlings were analyzed 10 d after nicosulfuron treatment at 5th leaf stage in this greenhouse research. The results showed that biological mass, total chlorophyll, chlorophyll a, and carotenoids content, photosynthetic rate, stomatal conductance, PS II maximum quantum yield, PS II effective quantum yield, PS II electron transport rate, photochemical quenching, maximal P₇₀₀ change, photochemical quantum yield of PS I, and PS I electron transport rate decreased with increasing herbicide concentrations, whereas initial fluorescence, quantum yield of non-regulated energy dissipation in PS II and quantum yield of non-photochemical energy dissipation due to acceptor side limitation in PS I increased. It suggests that nicosulfuron ≥1 mg L−1 causes the damage of chloroplast, PS II and PS I structure. Electron transport limitations in PS I receptor side, and blocked dark reaction process may be the main cause of the significantly inhibited growth and decreased photosynthetic rate of Radix Isatidis seedlings.     

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。     

Group 1 and 2 LEA protein expression correlates with a decrease in water stress induced protein aggregation in horsegram during germination and seedling growth

Plants produce an array of proteins as a part of a global response to protect the cell metabolism when they grow under environmental conditions such as drought and salinity that generate reduced water potential. The synthesis of hydrophilic proteins is a major part of the response to water deficit conditions. An increased expression of LEA proteins is thought to be one of the primary lines of defense to prevent the loss of intercellular water during adverse conditions. These LEA proteins are known to prevent aggregation of a wide range of other proteins. In this study we report the water stress induced protein aggregation and its abrogation followed by expression of group 1 and group 2 LEA proteins of water soluble proteomes in horsegram. Water stress caused an increased protein aggregation with magnitude and duration of stress in horsegram seedlings. Tissue-specific expression of LEA 1 protein decreased in the embryonic axis when compared to cotyledons in 24 h stressed seedlings. We found no cross reaction of LEA 1 with proteome of 48 h stressed embryonic axis and 72 h stressed root and shoot samples. However, LEA 2 antibodies were cross reacted with four polypeptides with different molecular mass in shoot tissue samples and found no reaction with root proteome as evidenced from immuno-blot analysis. The role of LEA proteins in relation to protein aggregation during water stressed conditions was discussed.     

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.     

Overexpression of a maize dehydrin gene, <Emphasis Type="Italic">ZmDHN2b</Emphasis>, in tobacco enhances tolerance to low temperature

Dehydrins, a subfamily of group 2 LEA proteins, are intrinsically unstructured plant proteins that accumulate in the late stages of seed development and in vegetative tissues subjected to water deficit, salinity, low temperature, or abscisic acid treatment. In this study, we isolated and characterized ZmDHN2b, a maize dehydrin gene. The genomic organization of the ZmDHN2b gene and its expression in maize seedlings were analyzed. To investigate the function of ZmDHN2b, we generated transgenic tobacco plants constitutively overexpressing ZmDHN2b. Ectopic expression of ZmDHN2b in tobacco accelerated seed germination and seedling growth at 15°C. Furthermore, ZmDHN2b-overexpressing lines had lower levels of cold-induced malondialdehyde and less electrolyte leakage than wild-type tobacco at 4°C. These results demonstrated that ZmDHN2b was involved in plant responses to low temperature.     

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。