盐胁迫下植物基因的表达与基因工程研究进展

在各种环境胁迫中,盐胁迫是造成作物减产的严重环境因素之一。随着植物分子生物学快速发展,植物耐盐性研究已深入到耐盐相关基因的克隆、基因的结构分析以及基因表达领域。文中就与植物耐盐性密切相关的小分子渗透物质、晚期胚胎发生富集蛋白(LEA)、通道蛋白、盐胁迫相关基因、信号传导基因和转录因子研究作了综述。同时对植物耐盐性研究作了简单的展望。     

植物耐盐相关基因克隆的研究进展

随着植物分子生物学快速发展,植物耐盐性研究已深入到耐盐相关基因的克隆、基因的结构分析以及基因表达特性等领域.目前,耐盐相关基因的克隆工作进行的如火如荼,有很多植物的耐盐基因已经被克隆,这些已克隆的耐盐相关基因涉及盐胁迫信号传导、基因表达的调控因子、渗透调节物质、胚胎发育晚期丰富蛋白LEA(Late-embryogensiS-abundant)等,本文就盐胁迫涉及的信号传导基因、基因表达调控因子等的克隆研究进展作一简要概述.     

红梨PybHLH基因过表达提高烟草NaCl抗性的研究

为探究过表达云南红梨bHLH转录因子对烟草抗盐性的影响,从红梨红色果皮中分离了bHLH转录因子基因PybHLH.亚细胞定位表明PybHLH蛋白定位于细胞核.以转基因PybHLH烟草和野生型烟草为材料,进行了NaCl胁迫对转基因PybHLH烟草生理生化影响研究及其相关酶基因的表达分析.表明PybHLH转基因烟草具有一定的耐盐性,一方面表现为随着盐胁迫时间延长,PybHLH转基因烟草中总可溶性糖、可溶性总蛋白和游离脯氨酸含量的增加,H2O2含量降低;另一方面表现为脯氨酸生物合成关键酶基因P5CS、抗氧化相关基因MnSOD、CuZn-SOD和POD、胁迫相关基因HSP和HSP cherpron和ABA抗盐信号途径基因NAC等均呈上调表达趋势.PybHLH的过表达提高了烟草的耐盐性,这将为进一步研究植物的耐盐机制及耐盐植物新品种的开发奠定基础.     

过表达秋茄C2H2型锌指蛋白基因KcZFP提高烟草耐盐性

基因转录调节是植物对非生物胁迫适应机制的一个重要方面,转录调节因子在胁迫信号转导途径中调节下游基因的表达,在建立植物对胁迫适应性过程中起到重要作用.锌指蛋白是功能多样的转录调节因子蛋白家族,家族成员在植物响应非生物胁迫方面扮演着重要角色.本研究以秋茄C2H2型锌指蛋白编码基因KcZFP为目的基因,在烟草中过表达KcZFP,分析C2H2型锌指蛋白在植物耐盐性中的作用.研究结果显示:转基因株系中,KcZFP表达量显著提高.过表达KcZFP的烟草植株的耐盐性明显提高,在200 mmol/L NaCl处理的条件下,KcZFP过表达烟草中脯氨酸水平远高于野生型植株.对光合作用参数比较分析显示,在KcZFP过表达植株中净光合速率受盐胁迫的影响小于野生型植株,光合系统在一定程度上得到了保护.研究结果说明KcZFP作为转录调节因子参与了植物的渗透调节,对植物的耐盐性具有贡献.     

植物耐盐基因工程研究进展

盐害是影响植物生长和作物产量的主要因素之一。用于提高植物耐盐性的基因工程方法很多,最常见的就是在植物中过量表达抗盐相关的功能基因,包括植物信号传导蛋白基因、植物离子通道蛋白基因和合成小分子渗透剂的酶基因等。归纳了近年来植物耐盐基因工程的研究进展,并展望了植物耐盐基因工程的研究前景。     

胡杨谷胱甘肽过氧化物酶PeGPX基因的克隆及转化植株耐盐性分析

胡杨(Populus euphratica Oliv.)具有极强抗盐碱能力。本实验室前期胡杨微阵列芯片数据结果显示:盐胁迫下,胡杨谷胱甘肽过氧化物酶基因(PeGPX)的转录上调,暗示该基因可能对胡杨耐盐性具有一定的作用。为分析 GPX 对植物耐盐性的贡献,本研究以胡杨为材料,利用 RT-PCR 方法克隆了胡杨谷胱甘肽过氧化物酶PeGPX基因,并在烟草中过量表达该基因,以分析谷胱甘肽过氧化物酶活性与植物耐盐性的关系。研究结果显示,实验中克隆的 cDNA (PeGPX)编码谷胱甘肽过氧化物酶,其 ORF 为 693 bp,其蛋白由 231 个氨基酸编码。过量表达 PeGPX 基因的烟草与野生型烟草的耐盐性实验结果显示,野生型烟草植株在加 NaCl(200 mmol/L)的 MS 培养基中生长 15 d 后,无明显的长高,且不长根;而转基因烟草在同样的加盐培养基上,生长基本没有受到抑制,植株生长状态良好,并且能够长根。光合数据显示,在盐胁迫下过量表达 PeGPX 基因烟草的净光合速率受到影响明显小于野生型烟草的净光合速率。酶活数据显示,转基因株系 GPX 酶活与野生型的相比在盐胁迫下活性有非常显著的提高。我们的研究结果说明:过表达 PeGPX 基因使得烟草的耐盐性得到显著提高,这对深入研究PeGPX基因在胡杨耐盐机制中的作用具有重要的意义。高,这对深入研究PeGPX基因在胡杨耐盐机制中的作用具有重要的意义。     

向日葵E3泛素连接酶基因的分析定位和表达鉴定

该研究利用前期获得的向日葵耐盐相关基因E3泛素连接酶基因序列(HERC2),构建瞬时表达载体Cam-35S-HERC2-GFP,采用基因枪法转化洋葱表皮细胞进行亚细胞定位;采用RT-PCR技术,分析盐胁迫下HERC2在耐盐品种P50和盐敏感品种P29根、下胚轴和叶中的表达差异;构建HERC2植物表达载体pPZP221-HERC2,采用农杆菌介导法将HERC2导入烟草,进行耐盐功能验证。结果表明:(1)HERC2蛋白定位在细胞膜、细胞质和细胞核中。(2)受到NaCl胁迫后,HERC2基因在耐盐品种P50和盐敏感品种P29中均上调表达,但耐盐品种中的表达量较高。(3)HERC2基因的表达,能够提高转基因烟草的耐盐性。该研究结果为进一步解析向日葵对盐胁迫的响应机制,以及耐盐新品种的选育奠定了基础。     

无苞芥锌指蛋白基因OpZFP提高烟草耐盐性的研究

旨在研究C2H2型锌指蛋白在植物生长发育、非生物胁迫信号转导过程中的作用。前期从新疆无苞芥中克隆的一个单锌指基因Op ZFP,利用叶盘法将Op ZFP基因转入普通烟草中。半定量RT-PCR表明,在转基因植株中Op ZFP基因能够高效表达。烟草耐盐性分析显示,在高盐胁迫下,转基因植株的根长要长于野生型植株,且转基因烟草丙二醛(MDA)的含量要明显低于野生型植株;并且高盐胁迫处理,野生型烟草离体叶片叶绿素降解率高于转基因植株。这些结果表明,过量表达Op ZFP的转基因植株可以提高植物对盐胁迫的抗性。     

过表达TaLEA1和TaLEA2基因提高转基因拟南芥的耐盐性

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。耐盐基因挖掘对作物耐盐育种非常重要。LEA蛋白家族是一个多基因家族,在植物应对非生物胁迫中发挥重要作用。本课题组前期研究阐明小麦TaLEA1基因在拟南芥中过表达可以提高转基因植物的耐盐性和抗旱性。本研究系统分析了小麦TaLEA2基因表达蛋白的理化性质、基因表达模式及启动子功能区域,并在拟南芥中过表达TaLEA2基因及共表达TaLEA1和TaLEA2基因,分析TaLEA2基因的抗逆功能及2个LEA基因的抗逆效果。结果表明,TaLEA2基因的表达产物属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。TaLEA2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TaLEA2基因,或过表达TaLEA1和TaLEA2基因都能够提高转基因拟南芥的耐盐性和抗旱性,转基因株系的种子萌发率、根长及叶绿素含量显著高于野生型,且双基因过表达的转基因植物的抗逆能力高于单个基因过表达株系。本研究结果为LEA基因抗逆机理的研究和多基因共转提高植物抗逆性提供了重要信息。     

费尔干猪毛菜病程相关蛋白基因（SfPR-1）的克隆及在盐胁迫下的表达分析

利用抑制消减杂交法从藜科猪毛菜属盐生植物费尔干猪毛菜（Salsola ferganica）中分离得到了一个盐胁迫响应的cDNA片段,结合SMARTTMRACE技术获得了费尔干猪毛菜病程相关蛋白基因的cDNA,命名该基因为SfPR-1（GenBank登录号：JQ670917）。序列分析表明,SfPR-1长817 bp,含有501 bp的阅读框、65 bp的5＇-UTR和251 bp的3＇-UTR,编码166个氨基酸,分子质量为18.01 kD,理论等电点为9.37。通过BLAST同源序列比对分析,结果显示该基因编码的蛋白与已知甜菜、拟南芥、烟草及玉米的病程相关蛋白PR-1同源性分别为73.6%、57.8%、55.5%和53.9%,且具有PR-1家族特有的6个半胱氨酸保守结构域。半定量RT-PCR和实时荧光定量RT-qPCR分析表明,该基因在盐胁迫后表达呈明显上调,初步推测病程相关蛋白基因SfPR-1可能与费尔干猪毛菜的耐盐性相关。     

Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803

The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.     

枯草芽孢杆菌耐盐突变株proA基因克隆及proBA基因渗透压调节功能研究

利用TaKaRaLAPCRTM试剂盒扩增枯草芽孢杆菌 931 5 1耐盐突变株proA基因的未知下游序列。根据测序结果 ,设计引物 ,克隆出发菌株和突变株全长proBA基因。将出发菌株和突变株的proBA基因分别转化大肠杆菌JM83(proBA- ) ,均能够与其功能互补。SDS PAGE分析其表达产物 ,有两条分子量分别约为 4 0kD和 4 5kD的新蛋白带出现。测定 4种转化子 (分别含有出发菌株和突变株proB基因的大肠杆菌 1 1 2 5 2转化子及proBA基因的大肠杆菌JM83转化子 )的耐盐能力。发现含有突变株proB或proBA基因转化子的耐盐能力 ,均比相应的含有出发菌株proB或proBA基因的转化子高。另外含有出发菌株和突变株的proBA基因转化子的耐盐能力 ,也均比相应的仅含proB基因的转化子高 ,表明枯草芽孢杆菌的ProA比大肠杆菌的ProA更为有效。测定所有JM83转化子胞内自由脯氨酸 ,发现其含量随盐浓度的上升而提高 ,其中含突变菌株proBA基因的转化子提高更为显著     

Cloning and Expression Analysis of Salt Responsive Gene from Kandelia candel

Identification of gene expression patterns in mangroves grown under salinity will help to reveal the molecular mechanisms of salt tolerance. Here, 10 cDNAs of genes were isolated from Kandelia candel and identified by representational difference analysis of cDNA (cDNA RDA) under different NaCl concentrations. Of five genes expressed preferentially under salt condition, two were unknown, three were two kinds of low molecular mass heat-shock proteins (sHSPs) and ADP-ribosylation factor, respectively. The expressions of other five genes were repressed under NaCl stress, two encoded cyclophilins, three were tonoplast intrinsic protein, early light-induced protein and 60S ribosomal protein, respectively.     

过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力

CBF/DREB是一类植物中特有的转录因子,在植物抵抗逆境胁迫过程中发挥重要功能。本研究从陆地棉(Gossypium hirsutum L.)Coker 312中克隆获得1个棉花CBF/DREB基因,命名为Gh CBF2,该基因编码一个由216个氨基酸组成的CBF蛋白。序列分析结果显示,Gh CBF2与其他植物的CBF蛋白类似,含有AP2转录因子典型的保守结构域。干旱或高盐胁迫处理明显增加了Gh CBF2基因的表达量。亚细胞定位分析结果发现Gh CBF2定位在细胞核中。将Gh CBF2基因构建到由35S启动子调控的植物表达载体p MD上并转化拟南芥(Arabidopsis thaliana L.),结果表明,在干旱和盐胁迫条件下,过量表达Gh CBF2基因拟南芥的成活率显著高于野生型,并且游离脯氨酸和可溶性糖含量也高于野生型,说明转Gh CBF2基因提高了拟南芥的耐盐抗旱能力。采用实时荧光定量PCR方法分析胁迫相关标记基因COR15A、RD29A和ERD6的表达情况,结果显示转基因株系中的表达量显著高于野生型,说明Gh CBF2参与调控拟南芥干旱和盐胁迫相关基因的表达。     

植物盐胁迫应答转录因子的研究进展

盐胁迫会导致植物受到初级的渗透胁迫和离子毒害以及次级的氧化胁迫和营养胁迫,严重制约了农业生产.植物盐胁迫应答转录因子能够通过调节下游靶基因的表达减轻盐胁迫对植物造成的伤害.文中基于土壤盐渍化及其对植物的危害、转录因子在植物盐胁迫信号转导网络中的中枢调节作用,综述了盐胁迫应答转录因子参与的盐胁迫信号转导途径、通过形成同源...     

Genome-Wide Identification of the F-box Gene Family and Expression Analysis under Drought and Salt Stress in Barley

The F-box protein-encoding gene family plays an essential role in plant stress resistance. In present study, 126 non-redundant F-box genes were identified in barley (Hordeum vulgare L., Hv). The corresponding proteins contained 165– 887 amino acid residues and all were amphiphilic, except 5 proteins. Phylogenetic analysis of F-box protein sequences in barley and stress-related F-box protein sequences in wheat and Arabidopsis thaliana (At) was used to classify barley F-box genes are divided into 9 subfamilies (A–I). A structure-based sequence alignment demonstrated that F-box proteins were highly conserved with a total of 10 conserved motifs. In total, 124 F-box genes were unevenly distributed on 7 chromosomes; another 2 genes have not been anchored yet. The gene structure analysis revealed high variability in the number of exons and introns in F-box genes. Comprehensive analysis of expression profiles and phylogenetic tree analysis, a total of 12 F-box genes that may be related to stress tolerance in barley were screened. Of the 12 detected F-box genes, 8 and 10 were upregulated after drought and salt stress treatments, respectively, using quantitative real-time polymerase chain reaction (qRT-PCR). This study is the first systematic analysis conducted on the F-box gene family in barley, which is of great importance for clarifying this family’s bioinformatic characteristics and elucidating its function in barley stress resistance. These results will serve as a theoretical reference for subsequent research on molecular regulation mechanisms, genetic breeding, and improvement.     

Functional Characterization of Maize C<Subscript>2</Subscript>H<Subscript>2</Subscript> Zinc-Finger Gene Family

Plant C2H2-type zinc finger proteins (ZFPs) play essential roles in developmental control and stress responses. The whole complement of ZFP genes has been identified in Arabidopsis and rice, while the genome-scale identification and functional analysis of maize ZFPs is not yet reported. Hence, we performed a comprehensive analysis, including gene structure, chromosome location, duplicated event, selective pressure, phylogeny, gene ontology annotation, and expression profiling under developmental stages and abiotic stresses. Phylogenetic analyses suggested that the ZmZFP gene family can be grouped into three classes (A, B, and C). The analysis of differential gene expression in different developmental stages and stress treatments (drought, salt, and cold) was conducted based on microarray and RNA-seq data. A total of 99.05 % (209 genes) of the total ZmZFP genes (211 genes) were detected in 60 different tissues in microarray data. Under drought stress, 13 differentially expressed genes were found in leaf, of which 7 and 6 genes were up-regulated and down-regulated, respectively. For salt stress, crown root (CR), primary root (PR) and seed root (SR) each had one significantly elevated gene, while 2, 1, and 7 genes were obviously down-regulated in CR, PR and SR, respectively. Additionally, 8 and 3 genes were significantly up-regulated and down-regulated, respectively, in the cold-tolerant line ETH-DH7. This study will lay the foundation for understanding the roles of ZFPs in maize growth and stress resistance, contributing to the molecular breeding of maize for food.     

基因芯片技术研究柽柳NaHCO3胁迫下基因的表达

运用基因芯片技术研究了NaHCO3胁迫下柽柳(Tamarix androssowii)基因的表达.将Cy5和Cy3两种荧光染料分别标记在NaHCO3处理和对照的柽柳cDNA上,将两种荧光探针混合,与载有柽柳基因的高密度芯片进行杂交并用芯片扫描系统进行扫描,通过Cy5与Cy3信号强度比值的计算研究基因的差异表达.共获得了89个差异表达的基因,其中,27个下调表达,62个上调表达.BlastX分析表明这些基因按功能可以分为光合作用、活性氧清除、渗透调节、信号传导与表达调控、代谢、发育相关、核糖体蛋白、蛋白质的分解与再生、转运类蛋白、水通道蛋白等几大类别.同时,发现了一些与盐胁迫相关的功能未知基因或未有任何功能信息的基因,这些基因可能在柽柳抗盐过程中具有重要作用.揭示了柽柳的抗盐胁迫涉及的几种重要途径,并获得了NaHCO3胁迫前后柽柳基因表达谱.