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。     

沙冬青AmNAC3转录因子基因在抗旱性和抗寒性中的功能鉴定

为了研究强抗逆植物沙冬青Am NAC3转录因子基因在抗旱性和抗寒性中的功能,首先利用半定量RT-PCR方法对该基因进行了表达分析。结果表明,在室内培养的沙冬青幼苗中,Am NAC3有一定量的基础表达,在干旱胁迫下其转录水平明显上调,而在低温胁迫下其表达上调较弱。然后利用5'RACE技术获得该基因的5'端序列及全长cDNA序列,并利用RT-PCR方法克隆到其全长编码区(846bp)。将编码区片段构建到植物表达载体上,利用农杆菌介导法获得转基因拟南芥。进一步分析表明,转基因拟南芥对于干旱和低温胁迫的抗性表型与野生型无明显差异,但其离体叶片的失水率和气孔开度均大于野生型。此外,转基因幼苗中气孔开闭相关基因ABI1和ABI2的表达量降低。这些结果表明,Am NAC3可能主要在响应干旱胁迫和调节气孔开闭及叶片保水性中发挥功能,而在抵抗低温胁迫中无明显作用。     

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.     

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。     

珙桐中一个与低温相关基因的克隆及其表达研究

低温诱导膜蛋白是由低温诱导表达蛋白基因编码的一类疏水性蛋白,在植物抵御寒冷环境时起着一定的保护作用。从珙桐cDNA文库中获得一个未知基因（DiRCI）,该基因长539bp,其中包括174bp的开放阅读框,92bp的5′末端非编码区和273bp的3′末端非编码区,编码57个氨基酸残基蛋白,在氨基酸水平上同源性较高的是车前草的低温诱导膜蛋白（登入号：ACA66247.1）,其相似性为89.5%。半定量RT-PCR分析发现,25℃以上,该基因在珙桐各器官中基本上均未表达,但经8℃低温处理时,在成熟叶片、叶柄和成熟未萌发的种子中均有表达,但是在根中基本没有表达,进一步研究发现该基因在24h～48h内表达量增多并达到最高值,48h后其表达量逐渐减弱直至消失,说明该基因确实与低温诱导相关,从而初步推测该基因为低温诱导膜蛋白基因。该基因的克隆丰富和保存了珙桐基因资源,并为进一步研究冷胁迫的分子机制奠定了基础。     

柠条锦鸡儿CkLEA4基因克隆及表达分析

LEA蛋白是一种与植物抗逆相关的胚胎发育晚期丰富蛋白。该研究从已构建的柠条锦鸡儿干旱胁迫抑制削减杂交文库中筛选到1条LEA蛋白编码基因的部分序列,用RACE技术扩增得到该基因cDNA全长并对其进行了克隆。测序表明该基因cDNA长870bp,其中开放阅读框长510bp,编码169个氨基酸,推测蛋白分子量为17.03kD,等电点为9.3,是一种亲水蛋白。序列比对和系统进化分析表明,该基因属于LEA4基因家族成员,命名为CkLEA4。实时荧光定量PCR检测发现,CkLEA4基因在干旱、ABA和NaCl处理下均受到不同程度的诱导,说明CkLEA4基因可能与柠条锦鸡儿响应逆境胁迫有关。     

小麦TaDRLea3 2基因的克隆及胁迫诱导表达分析

胚胎发育后期丰富蛋白(late embryogenesis abundant protein,LEA蛋白) 是植物体中广泛存在的一类与渗透调节相关的家族蛋白,植物受非生物胁迫会大量表达。该研究采用同源克隆技术,从干旱诱导的小麦品种‘郑引1号’ (Triticum aestivum L.)中获得1个新的LEA3家族基因（ TaDRLea3 2）,该基因全长668 bp,编码区为570 bp,编码189个氨基酸。生物信息学分析表明该蛋白为亲水性蛋白,二级结构以α 螺旋为主,含有9个由11个氨基酸组成的保守结构域,为典型的LEA3蛋白,存在3个磷酸化位点,无信号肽结构域及跨膜结构域,可能定位于细胞质中;实时定量PCR结果表明, TaDRLea3 2基因受干旱、高盐、低温诱导表达,同时也受外源ABA诱导,推测 TaDRLea3 2为ABA依赖型 LEA3基因,以不同机制参与小麦对非生物胁迫的应答过程,为深入分析小麦LEA3家族蛋白的抗逆机制奠定了基础。     

沙冬青脯氨酸转运体基因的克隆及表达分析

通过RACE技术从沙冬青中克隆获得了脯氨酸转运体基因Am Pro T(Gen Bank登录号为KJ873133)。序列分析表明,Am Pro T基因开放阅读框为1 329 bp,编码442个氨基酸,预测蛋白质分子量为48.07 k D,等电点为9.32,含有11个跨膜区域,具有典型的脯氨酸转运蛋白的特征。进化分析显示,Am Pro T与大豆Pro T的相似性达到86%。荧光定量PCR分析表明,Am Pro T在地上部的表达量要显著高于地下部,在受到干旱、高盐、脱落酸胁迫后表达量呈上调趋势,推测Am Pro T可能在沙冬青响应干旱、盐等非生物胁迫过程中发挥作用。     

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.     

葡萄VvBAP1基因的克隆及表达特性分析

以抗性葡萄品种‘F-242’组培苗为材料, 利用同源克隆法克隆了葡萄VvBAP1基因。测序结果显示, VvBAP1扩增片段大小为531 bp, 可编码176个氨基酸序列。利用生物信息学分析VvBAP1基因编码的蛋白序列显示, 该蛋白分子量为19.43 kDa, 含有保守的钙离子依赖性的C2结构域; 等电点pI为9.42; 不稳定系数为37.09, 推测为稳定的亲水性蛋白; 含有多个丝氨酸/苏氨酸磷酸化位点。实时荧光定量PCR表明, 该基因在根茎叶中均有表达, 其中在叶片中表达量较高; 盐胁迫、低温等逆境因子及逆境相关的信号物质, 如水杨酸和一氧化氮均可诱导VvBAP1的表达, 其中低温对其表达量影响更为显著, 推测该基因参与了葡萄抵御逆境胁迫的过程, 尤其是与低温相关的过程。     

中间锦鸡儿DHN1基因克隆及表达分析

脱水素(DHNs, dehydrins)是LEA II亚家族蛋白,在种子发育后期大量积累,并受不同逆境胁迫处理诱导表达。在中间锦鸡儿干旱胁迫抑制性削减杂交文库中筛选到了一段 DHN1 序列,利用RACE技术克隆获得基因全长序列。序列比对分析表明, CiDHN1 具有开放阅读框891bp,起始密码子为ATG,终止密码子为TAG,编码297个氨基酸,含有5个Y片段和一个K片段,与CiDHN1相似性最高的为山茱萸科主教红端木(Cornus sericea),相似性为39%。系统进化分析显示CiDHN1单独聚为一支,推测该蛋白为新的功能未知蛋白。亚细胞定位结果表明CiDHN1定位在细胞质、质膜和细胞核。荧光定量PCR结果显示 CiDHN1 的表达受冷、脱水和NaCl等非生物胁迫的诱导。 CiDHN1 过量表达拟南芥后,其中表达量最强的株系对200 mmol/L的NaCl处理较为敏感。CiDHN1在中间锦鸡儿抵抗逆境胁迫中的功能有待于进一步研究。     

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。     

Heterologous expression of a <Emphasis Type="Italic">Ammopiptanthus mongolicus</Emphasis> late embryogenesis abundant protein gene (<Emphasis Type="Italic">AmLEA</Emphasis>) enhances <Emphasis Type="Italic">Escherichia coli</Emphasis> viability under cold and heat stress

A late embryogenesis abundant protein gene, AmLEA from Ammopiptanthus mongolicus, was introduced into Escherichia coli using the IMPACT™-TWIN system to analyze the possible function of AmLEA under heat and cold stresses. A fusion protein about 38 kD was expressed in E.coli cells harboring pTWIN-LEA after the induction of IPTG by SDS–PAGE analysis and the accumulation of the fusion protein peaked 3 h after IPTG addition when cultured at 37°C. Compared with control cells, the E. coli cells expressing AmLEA fusion protein showed improved chilling and heat resistence, illuminating the protein may play a protective role in cells under stress conditions. These results suggested the natively unstructured protein, similar to other members of LEA proteins, has high capacity for binding water and potential protective function against dehydration or action similar to the cold shock chaperones.     

Molecular characterization and functional analysis by heterologous expression in E. coli under diverse abiotic stresses for OsLEA5, the atypical hydrophobic LEA protein from Oryza sativa L

In this study, we report the molecular characterization and functional analysis of OsLEA5 gene, which belongs to the atypical late embryogenesis abundant (LEA) group 5C from Oryza sativa L. The cDNA of OsLEA5 contains a 456 bp ORF encoding a polypeptide of 151 amino acids with a calculated molecular mass of 16.5 kDa and a theoretical pI of 5.07. The OsLEA5 polypeptide 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_2 domain architecture and is highly hydrophobic, intrinsically ordered with largely β-sheet and specific amino acid composition and distribution. Real-time PCR analysis showed that OsLEA5 was expressed in different tissue organs during different development stages of rice. The expression levels of OsLEA5 in the roots and panicles of full ripe stage were dramatically increased. The results of stress tolerance and cell viability assay demonstrated that recombinant E. coli cells producing OsLEA5 fusion protein exhibited improved resistance against diverse abiotic stresses: high salinity, osmotic, freezing, heat, and UV radiation. The OsLEA5 protein confers stabilization of the LDH under different abiotic stresses, such as heating, freeze–thawing, and drying in vitro. The combined results indicated that OsLEA5 protein was a hydrophobic 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.     

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.     

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。     

Overexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli and provides enhanced osmotic stress tolerance and ABA sensitivity in Arabidopsis thaliana

Previous studies have shown that the late embryogenesis abundant (LEA) group 3 proteins significantly respond to changes in environmental conditions. However, reports that demonstrate their biological role, especially in Arabidopsis, are notably limited. This study examines the functional roles of the Arabidopsis LEA group 3 proteins AtLEA3-3 and AtLEA3-4 in abiotic stress and ABA treatments. Expression of AtLEA3-3 and AtLEA3-4 is upregulated by ABA, high salinity, and osmotic stress. Results on the ectopic expression of AtLEA3-3 and AtLEA3-4 in E. coli suggest that both proteins play important roles in resistance to cold stress. Overexpression of AtLEA3-3 in Arabidopsis (AtLEA3-3-OE) confers salt and osmotic stress tolerance that is characterized during germination and early seedling establishment. However, AtLEA3-3-OE lines show sensitivity to ABA treatment during early seedling development. These results suggest that accumulation of AtLEA3-3 mRNA and/or proteins may help heterologous ABA re-initiate second dormancy during seedling establishment. Analysis of yellow fluorescent fusion proteins localization shows that AtLEA3-3 and AtLEA3-4 are mainly distributed in the ER and that AtLEA3-3 also localizes in the nucleus, and in response to salt, mannitol, cold, or BFA treatments, the localization of AtLEA3-3 and AtLEA3-4 is altered and becomes more condensed. Protein translocalization may be a positive and effective strategy for responding to abiotic stresses. Taken together, these results suggest that AtLEA3-3 has an important function during seed germination and seedling development of Arabidopsis under abiotic stress conditions.     

A LEA 4 protein up-regulated by ABA is involved in drought response in maize roots

Late embryogenesis abundant (LEA) proteins are hydrophilic proteins that accumulate to high concentrations during the late stages of seeds development, which are integral to desiccation tolerance. LEA proteins also play a protective role under other abiotic stresses. We analyzed in silico a maize protein predicted to be highly hydrophilic and intrinsically disordered. This prediction was experimentally corroborated by solubility assays under denaturing conditions. Based on its amino acid sequence, we propose that this protein belongs to group four of the LEA proteins. The accumulation pattern of this protein was similar to that of dehydrins during the desiccation process that takes place during seed development. This protein was induced by exogenous abscisic acid in immature embryos, but during imbibition was down-regulated by gibberellins. It was also induced in maize roots under osmotic stress. So far, this is the first member of the LEA proteins belonging to group four to be characterized in maize, and it plays a role in the response to osmotic stress.     

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.