大豆GmNFYB1基因功能预测及表达分析

本研究对大豆Gm NFYB1(Glyma02g17310)进行基因功能和表达分析,利用Plant CARE分析启动子元件发现,在基因启动子序列中含有多个与ABA、抗旱、光反应相关的元件。在The Bio-Analytic Resource for Plant Biology数据库中分析了Gm NFYB1的同源基因At NFYB5在不同逆境胁迫和不同激素处理时的基因表达情况,表明该基因表达具有组织表达特异性,参与盐胁迫、渗透胁迫等反应。Gm NFYB1在大豆的q RT-PCR结果表明,该基因受ABA、Na Cl、PEG诱导上调表达,参与大豆抗逆反应。     

番茄转录辅激活子基因LeMBF1在非生物胁迫中的抗性分析

为了研究番茄转录辅激活子基因LeMBF1在非生物胁迫中的作用,以LeMBF1超表达转基因番茄和野生型番茄组培苗为材料,对其进行高温、机械损伤、低温、H2O2、干旱和ABA等非生物胁迫处理.半定量RT-PCR分析表明,LeMBF1在根、幼叶、成熟叶片、花和果实的不同时期均有表达,但在幼叶中表达量最高,成熟叶片和果实中表达量较低.高温处理后,下游防卫反应基因HSP90、Apx2、Zat、PR1在超表达番茄植株中的表达量明显高于野生型番茄植株,并且LeMBF1超表达番茄植株存活率较高.机械损伤可以明显的诱导LeMBF1基因表达;H2O2、干旱、低温、和ABA处理后,对下游防卫基因的表达量有不同程度的影响.以上结果表明LeMBF1基因参与了多种植物非生物胁迫防御反应,并对提高番茄的抗性有一定的作用.     

水曲柳中MYBL2基因表达特征分析

MYB转录因子家族是植物中最大的转录因子家族之一,参与植物生长、繁殖和代谢的各个时期,能通过多种方式参与植物抗逆生长。该文在水曲柳中克隆FmMYBL2基因,利用生物信息学分析其结构和表达特征,并构建FmMYBL2蛋白的系统进化树。对水曲柳幼苗进行低温胁迫、盐胁迫处理以及激素分子诱导处理(包括ABA、IAA、GA_3、JA、SA)。分别在0、1、3、6、12、24、48 h取样,利用实时荧光定量PCR对上述处理样品中FmMYBL2基因进行定量分析,并分析了FmMYBL2的时空表达特征。结果表明:(1)克隆得到的FmMYBL2基因全长为762 bp,编码253个氨基酸。(2) FmMYBL2蛋白是亲水性蛋白,氨基酸序列比对表明其与棉花同源关系较近。(3)荧光定量分析表明,FmMYBL2基因响应低温胁迫和盐胁迫,同时ABA、IAA、GA_3、JA、SA共同调控该基因表达。(4)在低温处理1 h、盐胁迫48 h时,虽然FmMYBL2基因表达量最高,激素诱导后表达量持续波动,但其能在短时间内迅速响应。(5) FmMYBL2基因在根、芽、花、种子中均有表达,雄花中的表达量最高。该研究结果为深入研究MYBL2基因功能和水曲柳抗逆生长的调控奠定基础。     

H2O2和ABA对干旱高温复合胁迫诱导的玉米叶片sHSPs表达的影响

以玉米脱落酸(ABA)缺失突变体vp5及其野生型Vp5的叶片为材料,分别采用ABA、碘化钾(H2O2清除剂)、钨酸钠(ABA抑制剂)预先处理,对干旱+高温复合胁迫下玉米叶片小热休克蛋白(sHSPs)基因表达进行研究,以确定H2O2和ABA对干旱+高温复合胁迫诱导的玉米叶片sHSPs基因表达的影响。结果显示:(1)与对照和干旱相比,高温、干旱+高温复合胁迫显著诱导了sHSP16.9、sHSP17.2、sHSP17.4、sHSP17.5、sHSP22和sHSP26等6种sHSPs的表达。(2)H2O2清除剂KI和ABA抑制剂钨酸钠预处理,仅略微抑制高温、干旱+高温复合胁迫诱导的6种sHSPs表达。(3)与未用100μmol/L ABA预处理的vp5相比,100μmol/L ABA预处理仅略微提高了高温、干旱+高温复合胁迫诱导的6种sHSPs的表达水平。研究表明,在干旱+高温复合胁迫条件下H2O2和ABA参与了干旱+高温复合胁迫诱导的玉米叶片sHSPs表达,但并无显著影响,暗示了H2O2和ABA不是干旱+高温复合胁迫诱导sHSPs表达的重要调控因子。     

2个慈竹bZIP基因的克隆、生物信息学分析及其诱导表达

从慈竹笋转录组数据库中筛选并克隆出2个bZIP基因（BebZIP2和BebZIP6）进行生物信息学分析。分析结果表明,它们编码序列长度分别为504和720 bp,编码167个和239个氨基酸,BebZIP2和BebZIP6属于bZIP相关蛋白,与水稻OsbZIP52/RISBZ5蛋白聚在一枝。组织表达分析表明,这2个慈竹BebZIP基因在慈竹笋、茎、展开叶和未展开叶等部位均有表达,属于组成型表达,同一基因在不同组织中的表达量差异较大,表达量的大小为未展开叶 > 展开叶 > 茎 > 笋。对慈竹幼苗进行ABA、NaCl和PEG6000非生物胁迫处理,结果表明,BebZIP2和BebZIP6对盐、干旱和ABA胁迫均具有不同程度的响应。     

水稻NAM基因家族的全基因组鉴定及表达分析（英文）

植物特异性转录因子NAM家族从属于NAC转录因子超家族,在植株生长发育、生理代谢以及应对各种胁迫反应中均发挥重要作用。该研究采用生物信息学方法鉴定水稻基因组中的NAM基因,分析其时空表达模式、亚细胞定位以及蛋白相互作用,并采用实时定量qRT-PCR方法分析不同外源激素(如SA、ABA和MeJA)以及非生物胁迫(包括干旱、盐和冷)处理下各NAM基因的表达特征,为进一步探索NAM基因在非生物胁迫中的功能和应激机制以及激素调控途径奠定基础。结果显示:(1)从水稻基因组中共鉴定出48个NAM基因,进化分析将其分为5个亚家族;NAM基因在水稻基因组中存在9对片段复制事件。(2)组织表达分析显示,NAM基因在水稻不同组织及发育时期表现特异性表达,特别是叶鞘、茎和节的生长过程中高表达,且大多数是核定位,并存在多种蛋白互作。(3)实时定量qRT-PCR表达分析显示,10个NAM基因在不同组织中均特异表达;大部分NAM基因在盐和干旱胁迫下表达上调,而在冷胁迫下表达降低;SA、ABA和MeJA处理均可显著改变各NAM基因的表达水平。研究表明,NAM基因在水稻生长发育、激素应答和非生物胁迫响应中具有重要作用。     

木薯MeNCED3基因克隆、结构变异及其表达分析

旨在揭示木薯MeNCED3基因在干旱等非生物胁迫应答中的作用。用RT-PCR的方法从木薯叶片中克隆MeNCED3基因,用MEGA软件构建进化树;用Dna SP软件分析基因结构变异,用荧光定量PCR技术分析MeNCED3在不同非生物胁迫下的表达特性。结果显示,从木薯中克隆了一个NCED基因MeNCED3,该基因具有1 803 bp的开放阅读框,编码600个氨基酸,含有NCED家族保守结构域。进化树分析表明,MeNCED3与杨树和杞柳中同源基因的亲缘关系较近,序列相似性分别达到83.9%和82.5%。基因结构变异发现,木薯野生种和栽培种之间共有8个错义突变,其中5个可能与MeNCED3的表达量有关。实时荧光定量PCR分析表明,MeNCED3在根中的表达量要远远高于叶片和茎。而且,MeNCED3的表达能被PEG、ABA和NaCl处理显著诱导。因此,MeNCED3在转录水平对木薯渗透胁迫、盐胁迫起调控作用,可作为候选基因进一步研究其在木薯抗旱中的功能。     

小麦E3泛素连接酶基因TaAIRP2-1B克隆及功能分析

干旱等非生物胁迫严重影响农作物生产。本研究克隆了小麦(Triticum aestivum L.)TaAIRP2-1B基因,探讨其对非生物胁迫的响应机制,为促进小麦抗旱性的遗传改良提供基因资源。组织特异性表达模式分析显示,TaAIRP2-1B基因在小麦抽穗期的各个组织中均有表达,在茎组织中的表达水平较高,而根系中的表达水平较低。非生物胁迫表达模式分析显示,Ta AIRP2-1B受ABA、PEG及冷胁迫诱导表达。过表达TaAIRP2-1B拟南芥在0.4μmol/L的ABA处理条件下,种子发芽率显著低于野生型,表明TaAIRP2-1B提高了拟南芥种子萌发期对ABA的敏感性。ABA处理抑制转基因和野生型拟南芥幼苗的根系生长,但转基因拟南芥受抑制程度显著高于野生型,表明TaAIRP2-1B提高了拟南芥幼苗对ABA的敏感性。转基因结果表明超表达TaAIRP2-1B增强了拟南芥的抗旱性,并且转基因株系的保水率显著高于野生型。总之,本研究发现小麦基因Ta AIRP2-1B参与了植物对非生物胁迫的应答,可能是通过ABA途径正向调控植物的抗旱性。     

模拟干旱胁迫下马铃薯StNCED1表达量及与ABA含量的相关性分析

干旱缺水是限制马铃薯产量和品质的关键因素之一。ABA是干旱胁迫应答基因调控网络中的重要组成;9-顺式-环氧类胡萝卜素双加氧酶(NCED)是植物ABA生物合成途径的关键限速酶,该基因的表达模式直接影响ABA代谢。但干旱胁迫下有关马铃薯NCED基因表达与激素、表型间的相关分析的研究尚少。本研究克隆了马铃薯NCED1基因的全长c DNA序列,检测了4个马铃薯种质材料在不同模拟干旱(PEG-6000)胁迫强度处理下NCED1基因表达量、ABA含量及根系长度间的相关性。结果表明,St NCED1全长2181 bp,包含一个1800 bp的完整开放读码框,编码599个氨基酸。5%PEG-6000和15%PEG-6000模拟干旱胁迫4周后,4个品种均表现出随胁迫强度的加重植株生长缓慢、矮小,根系长度显著降低。干旱敏感型材料早大白中根系长度变化最大,ABA含量显著高于其他3个种质材料。CIP478.9、star和米拉3份种质材料中St NCED1表达量与对照植株有显著差异,且随胁迫强度增加而增大;早大白中St NCED1表达量表现出先降低后升高趋势。模拟干旱胁迫下,ABA含量与St NCED1表达量之间呈现出正相关关系(R0.7)。研究结果为解析马铃薯响应干旱胁迫的调节机制及其在抗旱种质资源筛选中的应用提供了基础数据。     

水曲柳FmABI5基因非生物胁迫及信号诱导的表达模式分析

ABI5（abscisic acid-insensitive 5）蛋白是一个响应ABA信号的碱性亮氨酸拉链类（basic leucine zipper,bZIP）转录因子。揭示了水曲柳FmABI5在非生物胁迫下的表达特征,为该基因在水曲柳中代谢调控功能的研究奠定基础。获得了水曲柳ABI5基因的全长,命名为FmABI5,应用生物信息学软件分析了水曲柳FmABI5基因的分子结构特征,利用PEG、低温（4℃）及盐（NaCl）进行非生物胁迫处理,利用脱落酸（ABA）和赤霉素（GA₃）进行信号诱导,分析FmABI5的表达特征。生物信息学分析表明该基因全长1 455 bp,含有完整的开放阅读框,编码484个氨基酸。FmABI5为不稳定类亲水性蛋白,不存在信号肽,具有跨膜能力,α-螺旋、延伸链、无规则卷曲分布于整个蛋白。分子进化分析结果表明,水曲柳FmABI5基因与芝麻的遗传距离较近,说明其亲缘关系较近;与马铃薯、潘那利番茄、水茄与甜椒的遗传距离较远,说明其亲缘关系较远。非生物胁迫结果表明,FmABI5基因表达水平随非生物胁迫处理时间不同而上下波动,但在处理6、48和72 h后,FmABI5基因对3种非生物胁迫处理都上调表达,说明FmABI5基因对非生物胁迫具有响应。信号诱导结果表明,外源的ABA与GA共同调节了FmABI5基因的表达。     

Differential contributions to the transcriptome of duplicated genes in response to abiotic stresses in natural and synthetic polyploids

Polyploidy has occurred throughout plant evolution and can result in considerable changes to gene expression when it takes place and over evolutionary time. Little is known about the effects of abiotic stress conditions on duplicate gene expression patterns in polyploid plants. We examined the expression patterns of 60 duplicated genes in leaves, roots and cotyledons of allotetraploid Gossypium hirsutum in response to five abiotic stress treatments (heat, cold, drought, high salt and water submersion) using single-strand conformation polymorphism assays, and 20 genes in a synthetic allotetraploid. Over 70% of the genes showed stress-induced changes in the relative expression levels of the duplicates under one or more stress treatments with frequent variability among treatments. Twelve pairs showed opposite changes in expression levels in response to different abiotic stress treatments. Stress-induced expression changes occurred in the synthetic allopolyploid, but there was little correspondence in patterns between the natural and synthetic polyploids. Our results indicate that abiotic stress conditions can have considerable effects on duplicate gene expression in a polyploid, with the effects varying by gene, stress and organ type. Differential expression in response to environmental stresses may be a factor in the preservation of some duplicated genes in polyploids.     

Alfalfa Cellulose Synthase Gene Expression under Abiotic Stress: A Hitchhiker’s Guide to RT-qPCR Normalization

Abiotic stress represents a serious threat affecting both plant fitness and productivity. One of the promptest responses that plants trigger following abiotic stress is the differential expression of key genes, which enable to face the adverse conditions. It is accepted and shown that the cell wall senses and broadcasts the stress signal to the interior of the cell, by triggering a cascade of reactions leading to resistance. Therefore the study of wall-related genes is particularly relevant to understand the metabolic remodeling triggered by plants in response to exogenous stresses. Despite the agricultural and economical relevance of alfalfa (Medicago sativa L.), no study, to our knowledge, has addressed specifically the wall-related gene expression changes in response to exogenous stresses in this important crop, by monitoring the dynamics of wall biosynthetic gene expression. We here identify and analyze the expression profiles of nine cellulose synthases, together with other wall-related genes, in stems of alfalfa plants subjected to different abiotic stresses (cold, heat, salt stress) at various time points (e.g. 0, 24, 72 and 96 h). We identify 2 main responses for specific groups of genes, i.e. a salt/heat-induced and a cold/heat-repressed group of genes. Prior to this analysis we identified appropriate reference genes for expression analyses in alfalfa, by evaluating the stability of 10 candidates across different tissues (namely leaves, stems, roots), under the different abiotic stresses and time points chosen. The results obtained confirm an active role played by the cell wall in response to exogenous stimuli and constitute a step forward in delineating the complex pathways regulating the response of plants to abiotic stresses.     

Differential expression of ferritin genes in response to abiotic stresses and hormones in pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>)

In this study, the expression patterns of four ferritin genes (PpFer1, PpFer2, PpFer3, and PpFer4) in pear were investigated using quantitative real-time PCR. Analysis of tissue-specific expression revealed higher expression level of these genes in leaves than in other tested tissues. These ferritin genes were differentially expressed in response to various abiotic stresses and hormones treatments. The expression of ferritin wasn’t affected by Fe(III)-citrate treatment. Abscisic acid significantly enhanced the expression of all four ferritin genes, especially PpFer2, followed by N-benzylyminopurine, gibberellic acid, and indole-3-acetic acid. The expression peaks of PpFer1 and PpFer3 in leaves appeared at 6, 6, and 12 h, respectively, after pear plant was exposed to oxidative stress (5 mM H₂O₂), salt stress (200 mM NaCl), and heat stress (40°C). A significant increase in PpFer4 expression was detected at 6 h after salt stress or heat stress. The expression of ferritin genes was not altered by cold stress. These results suggested that ferritin genes might be functionally important in acclimation of pear to salt and oxidative stresses. Hormone treatments had no significant effect on expression of ferritin genes compared to abiotic stresses. This showed accumulation of ferritin genes could be operated by different transduction pathways under abiotic stresses and hormones treatments.     

Expression analysis of rice A20/AN1-type zinc finger genes and characterization of ZFP177 that contributes to temperature stress tolerance

The A20/AN1-type zinc finger protein family is conserved in animals and plants. Using human AWP1 protein as a query, we identified twelve A20/AN1-type zinc finger proteins in japonica rice. Most of these genes were constitutively expressed in leaves, roots, culms and spikes. Through microarray analysis, it was found that four genes (ZFP177, ZFP181, ZFP176, ZFP173), two genes (ZFP181 and ZFP176) and one gene (ZFP157) were significantly induced by cold, drought and H(2)O(2) treatments, respectively. Further expression analysis showed that ZFP177 was responsive to both cold and heat stresses, but down-regulated by salt. The subcellular localization assay indicated that ZFP177 was localized in cytoplasm in tobacco leaf and root cells. Yeast-one hybrid assay showed that ZFP177 lacked trans-activation potential in yeast cells. Overexpression of ZFP177 in tobacco conferred tolerance of transgenic plants to both low and high temperature stresses, but increased sensitivity to salt and drought stresses. Further we found expression levels of some stress-related genes were inhibited in ZFP177 transgenic plants. These results suggested that ZFP177 might play crucial but differential roles in plant responses to various abiotic stresses.     

拟南芥AtWRKY25、AtWRKY26和AtWRKY33在非生物胁迫条件下的表达分析

WRKY 转录因子家族在调控植物逆境诱导反应、生长发育及其信号转导等方面起着重要的分子生物学功能。文章采用Northern 杂交的方法, 对拟南芥3个WRKY基因进行表达谱分析。结果表明: AtWRKY25、AtWRKY26和AtWRKY33受多种非生物逆境因子(温度因子、高盐、渗透胁迫和激素脱落酸)的影响, 其中低温和高盐对AtWRKY25、AtWRKY26和AtWRKY33的诱导尤为明显, 表明这3个AtWRKY基因可能在响应环境信号方面起着一定的作用。作为序列相似性较高的AtWRKY25、AtWRKY26和AtWRKY33对一些胁迫因子的表达模式呈现一定的相似性; 但AtWRKY33受高温的抑制和低温的快速诱导, 与另外两个基因的表达模式不同, 推测它们对温度胁迫因子的反应存在差异。此外, 对启动子序列的生物信息学分析发现, 3个基因的启动子包含多个与非生物逆境反应相关的顺式作用元件。     

Molecular cloning and expression analysis of the replacement histone H3 gene of Italian ryegrass (Lolium multiflorum)

The replacement histone H3 gene and its 5'-flanking sequence were isolated from Italian ryegrass by polymerase chain reaction and inverse polymerase chain reaction, respectively. Expression analysis showed that this gene is constitutively expressed in the entire plant. The expression level in leaves was found to be significantly low when compared with that in other tissues. However, the gene expression level in leaves was increased by the treatment with abscisic acid and abiotic stresses such as cold, heat and high-salinity (NaCl). The motif search of the 5'-flanking sequence of the replacement histone H3 gene revealed the presence of several potential cis-acting elements that could respond to the above-mentioned abiotic stresses. In addition to defence-related elements, we also found type I and II-/III-like elements, which are highly conserved motifs in the 5'-regulatory sequence of plant histone genes that are expressed specifically during the S-phase. Experiments using transgenic Italian ryegrass plants proved that the isolated 5'-flanking sequence of the replacement histone H3 gene, which was fused to a beta-glucuronidase reporter gene, was fully functional for inducing gene expression under various abiotic stress conditions.