Identification and characterization of an expansin gene AsEXP1 associated with heat tolerance in C3 Agrostis grass species

Plant tolerance of heat stress involves various changes at physiological and molecular levels. The objective of this study was to examine the expression of a gene encoding expansin protein in relation to heat tolerance in two C(3) grass species and genotypes differing in heat tolerance. Heat-tolerant, thermal Agrostis scabra, adapted to high temperatures in geothermal areas in Yellowstone National Park, was subjected to 20 degrees C (control) or 40 degrees C (heat stress) for 7 d in a growth chamber. Differential display analysis identified that a gene, AsEXP1, encoding an expansin protein, was strongly up-regulated in leaves exposed to heat stress in thermal A. scabra. Virtual northern hybridization and RT-PCR confirmed that AsEXP1 was a heat-inducible gene in leaves. The expression of AsEXP1 was induced at 1 h of plant exposure to heat stress and reached the highest level of expression at 4 h of treatment. A 1.3 kb full-length cDNA of AsEXP1 was isolated, which encodes a 251 amino acid protein. Two ecotypes of thermal A. scabra and 10 genotypes of Agrostis stolonifera (creeping bentgrass), a widely used turfgrass species in cool climatic regions, varying in the level of heat tolerance, were exposed to 40 degrees C for 7 d to examine the level of AsEXP1 expression in relation to heat tolerance. Genetic variation in heat tolerance was evaluated by measuring cell membrane stability, photochemical efficiency, and leaf growth. RT-PCR analysis revealed that the level of AsEXP1 in different genotypes was positively correlated with the level of heat tolerance in both grass species. The results first identified a heat-related expansin gene in grass species and suggest that AsEXP1 may be useful as a molecular marker to select for heat-tolerant grass germplasm.     

Differential gene expression in shoots and roots under heat stress for a geothermal and non-thermal Agrostis grass species contrasting in heat tolerance

Identification of genes associated with heat tolerance is important for developing heat-tolerant plants. The objectives of this study were to compare genes differentially expressed in shoots and roots in two Agrostis grass species contrasting in heat tolerance under different temperature regimes and to identify up-regulated genes associated with heat-stress adaptation in Agrostis grass species. Heat-tolerant, thermal A. scabra, adapted to geothermal areas in Yellowstone National Park, and heat-sensitive A. stolonifera, used as a turf and forage grass in cool climatic regions, were exposed to 20 or 40 °C in growth chambers. Leaves and roots were sampled at 7 day of treatment to evaluate physiological responses to heat stress and to perform differential display analysis. Thermal A. scabara maintained significantly higher leaf chlorophyll content and root viability and lower electrolyte leakage (EL) following 7 day of heat stress, compared to A. stolonifera. Differential display analysis of leaf samples revealed that 21 gene fragments were down-regulated while 18 fragments were up-regulated in heat-stressed A. stolonifera. For thermal A. scabra, 38 gene fragments were down-regulated and 22 fragments were up-regulated under heat stress. Four gene fragments in roots were found to be up-regulated under heat stress. RT-PCR analysis confirmed that one gene fragment in leaves (AsL9) and two gene fragments in roots (AsR1 and AsR2) were expressed only in thermal A. scabra exposed to heat stress. These heat-inducible genes in thermal A. scabra may contribute to its superior ability to survive in chronically high-temperature soils in geothermal areas. Putative functional analysis with BLASTX found that most of the up-regulated genes in both species were involved in stress defense pathways or tolerance mechanisms, such as cell wall elasticity, secondary metabolism, regulatory functions, and protein synthesis.     

镍胁迫下安氏伪镖水蚤SSH文库的构建及铁蛋白cDNA的克隆与差异表达

为探讨镍胁迫下桡足类的分子响应机制,以安氏伪镖水蚤为研究对象,利用抑制性消减杂交(suppression subtractive hybridization,SSH)技术,构建了镍胁迫下安氏伪镖水蚤SSHcDNA文库,并随机挑取生长菌落140个克隆子,进行菌液PCR鉴定,计算文库重组率为98.6％,文库容量为1.12×106 cfu.将重组子测序,经BLAST一致性搜索比对分析发现,有一重组片段含有铁蛋白保守结构域,该片段大小为859 bp,连续编码170个氨基酸残基,Gen-Bank登录号为JK312601.半定量PCR证实,镍胁迫24 h后,安氏伪镖水蚤中铁蛋白表达显著上调.本文库的成功构建及铁蛋白cDNA片段的获得为进一步研究镍胁迫下桡足类的分子响应机制奠定了基础.     

利用抑制性扣除杂交技术克隆水稻磷饥饿诱导基因

磷素是植物生长所必需的重要元素。在缺磷环境中,植物能够调节自身的形态、生理生化和基因表达水平来适应环境的变化。为研究水稻(Oryzn sativa L.)耐低磷胁迫的分子机理,采用抑制性扣除杂交技术(SSH)构建磷饥饿诱导的水稻根系扣除cDNA文库。通过文库筛选和测序获得18个已知基因和47个功能未知基因。这些基因参与了不同的代谢过程,包括磷吸收和转运、信号传导、蛋白质合成和降解、碳水化合物代谢和胁迫反应。Northern杂交结果表明,在磷饥饿胁迫下这些基因呈现不同的表达模式,并且不同代谢过程中的基因对磷饥饿有着不同的反应。     

Root respiratory characteristics associated with plant adaptation to high soil temperature for geothermal and turf-type Agrostis species

Respiration is a major avenue of carbohydrates loss. The objective of the present study was to examine root respiratory characteristics associated with root tolerance to high soil temperature for two Agrostis species: thermal Agrostis scabra, a species adapted to high-temperature soils in geothermal areas in Yellowstone National Park, and two cultivars ('L-93' and 'Penncross') of a cool-season turfgrass species, A. stolonifera (creeping bentgrass), that differ in their heat sensitivity. Roots of thermal A. scabra and both creeping bentgrass cultivars were exposed to high (37 degrees C) or low soil temperature (20 degrees C). Total root respiration rate and specific respiratory costs for maintenance and ion uptake increased with increasing soil temperatures in both species. The increases in root respiratory rate and costs for maintenance and ion uptake were less pronounced for A. scabra than for both creeping bentgrass cultivars (e.g. respiration rate increased by 50% for A. scabra upon exposure to high temperature for 28 d, as compared with 99% and 107% in 'L-93' and 'Penncross', respectively). Roots of A. scabra exhibited higher tolerance to high soil temperature than creeping bentgrass, as manifested by smaller decreases in relative growth rate, cell membrane stability, maximum root length, and nitrate uptake under high soil temperature. The results suggest that acclimation of respiratory carbon metabolism plays an important role in root survival of Agrostis species under high soil temperatures, particularly for the thermal grass adaptation to chronically high soil temperatures. The ability of roots to tolerate high soil temperatures could be related to the capacity to control respiratory rates and increase respiratory efficiency by lowering maintenance and ion uptake costs.     

Cloning and characterization of a salt stress-inducible small GTPase gene from the model grass species Lolium temulentum

A gene encoding a small guanosine triphosphate (GTP)-binding protein (smGTP) related to the Rab2 gene family of GTPases was identified during the analysis of a salt stress suppression subtractive hybridization (SSH) expression library from the model grass species Lolium temulentum L. (Darnel ryegrass). The smGTP gene was found to have a low-level constitutive expression and was strongly induced by salt stress in root, crown and leaf tissues. The expression pattern of the smGTP gene was compared against two additional stress genes identified in the SSH expression library, the well-characterized dehydration stress tolerance gene, delta 1-pyrroline-5-carboxylate synthetase (P5CS) encoding for a key enzyme in proline biosynthesis, and the cold response gene COR413. The genes were analyzed for their response to salinity as well as their responses to 7 different forms of abiotic stress in L. temulentum plants. The smGTP gene displayed an expression pattern similar to the P5CS gene, suggesting a role in dehydration stress. In contrast, the COR413 gene was found to be up-regulated in response to all stresses tested and has utility as a general stress marker in grass plants.     

血管紧张素Ⅱ诱导心肌成纤维细胞表达上调基因的分离和鉴定

为了对成年大鼠心肌成纤维细胞（cardiac fibroblasts,CF）受血管紧张素Ⅱ（angiotensin Ⅱ,AngⅡ）刺激后上调基因表达谱进行筛选及分析,以受AngⅡ刺激CF为实验方,未刺激CF为驱动方,进行抑制消减杂交（suppression subtractive hybridization,SSH）,建立消减cDNA文库。经斑点杂交筛选文库后将表达变化显著的部分阳性克隆测序及同源性分析,共获得19个上调表达的基因,分别与细胞外基质、细胞周期、胞内信号转导、细胞骨架及细胞代谢等功能相关,并克隆到7个新的基因表达序列标签（expressed sequence tags,EST）。我们的数据证实了SSH可以有效地克隆成年大鼠CF受AngⅡ刺激后上调表达基因,对这些基因的研究将有助于阐明心肌重塑的分子机制。     

Identification of genes differentially expressed in cauliflower associated with resistance to <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">campestris</Emphasis>

Black rot, caused by Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc), is one of the most damaging diseases of cauliflower and other crucifers. In order to investigate the molecular resistance mechanisms and to find the genes related to black rot resistance in cauliflower, a suppression subtractive hybridization (SSH) cDNA library was constructed using resistant line C712 and its susceptible near-isogenic line C731 as tester and driver, respectively. A total of 280 clones were obtained from the library by reverse northern blotting. Sequencing analysis and homology searching showed that these clones represent 202 unique sequences. The library included many defense/disease-resistant related genes, such as plant defensin gene PDF1.2, lipid transfer protein, thioredoxin h. Gene expression profiles of 12 genes corresponding to different functional categories were monitored by real-time RT-PCR. The results showed that the expression induction of these genes in the susceptible line C712 in response to Xcc was quicker and more intense, while in C731 the reaction was delayed and limited. Our results imply that these up-regulated genes might be involved in cauliflower responses against Xcc infection. Information obtained from this study could be used to understand the molecular mechanisms of disease response in cauliflower under Xcc stress.     

锌胁迫下小麦SSH文库的构建及初步分析

Zn是植物必需的微量元素,同时也是近年来造成环境污染的重金属元素之一.为了从基因表达水平揭示小麦Zn胁迫响应的分子机制,本研究利用抑制差减杂交(suppression subtractive hybridization,SSH)技术构建了Zn胁迫（0.5 mmol/L,1 mmol/L）下小麦的正反向SSH文库.从正反向文库中随机挑选阳性单克隆,并利用通用引物T7/Sp6对其进行验证. 结果显示,正反库中分别获得307和821个EST序列,其片段长度在200～1 000 bp之间,它们反映了Zn胁迫下特异响应的基因.利用BLASTn和BLASTx将这些EST序列进行比对分析,在正、反库中分别筛选出221和641个uniESTs,其中751个uniESTs被注释（包括正库中193个和反库中558个）.这些序列的功能主要涉及信号转导、抗氧化防御、转录与翻译、物质运输、核糖体结构、能量代谢,以及一些功能未知的基因.     

干旱胁迫下刚毛柽柳消减文库的构建及分析

以干旱胁迫下的刚毛柽柳根部组织cDNA为tester,正常生长的刚毛柽柳根部组织cDNA为driver,利用抑制性消减杂交技术(SSH)构建了干旱胁迫下刚毛柽柳根部组织的消减文库。文库克隆的重组率为95%,插入片段大部分集中在250~600 bp之间。通过对文库阳性克隆的随机测序,获得了如Mn-SOD、myb相关蛋白、锌指蛋白等17种与干旱胁迫相关的基因,它们涉及了植物的渗透调节、信号传递、转录调控、活性氧清除等方面。所得EST序列已被GenBank收录。实验为抗逆基因克隆和系统研究干旱胁迫下柽柳根部基因的表达奠定了基础。     

盐胁迫下西伯利亚蓼茎叶正反消减文库的构建及初步分析

正向文库以3% NaHCO3胁迫48 h的材料(茎、叶)为实验方(tester), 以未胁迫的材料(茎、叶)为消减方(driver); 在负向文库中, 以未胁迫的材料(茎、叶)为实验方(tester), 以3% NaHCO3胁迫48 h的材料(茎、叶)为消减方(driver), 利用抑制性消减杂交技术(suppression subtractive hybridization, SSH)构建了3% NaHCO3胁迫下西伯利亚蓼茎叶正反消减文库。从文库中共获得2 282条有效EST序列, 其中从茎正向消减文库中获得598条, 从茎负向消减文库中获得490条, 从叶正向消减文库中获得627条, 从叶负向消减文库中获得567条。经BlastX序列比对后, 通过MIPs方法对EST功能进行分类并对茎叶正反消减文库做了比较, 其中茎与叶部除细胞救援与防御、转运组件中变化趋势相同外, 在代谢、能量、光合作用、蛋白合成、转录和信号传导等方面均表现为相反趋势, 另外通过荧光定量RT-PCR对12个潜在与盐胁迫相关基因进行定量分析, 结果显示12个基因在未经胁迫与盐胁迫处理后的西伯利亚蓼叶与茎部有着很大差异, 说明叶与茎部在NaHCO3条件下可能具有不同应答机制与分工, 这有助于进一步理解西伯利亚蓼分子耐盐机制。     

Short-term and long-term root respiratory acclimation to elevated temperatures associated with root thermotolerance for two Agrostis grass species

This study was designed to investigate whether thermotolerant roots exhibit respiratory acclimation to elevated temperatures. Root respiratory acclimation traits in response to increasing temperatures were compared between two Agrostis species contrasting in heat tolerance: thermal A. scabra and heat-sensitive A. stolonifera. Roots of both species were exposed to 17, 27, or 37 degrees C. Root RGR declined with increasing temperatures from 17 degrees C to 37 degrees C in both species; however, root growth of A. scabra maintained a significantly higher RGR than A. stolonifera at 27 degrees C or 37 degrees C. A. scabra exhibited a significantly higher respiration acclimation potential to elevated temperatures, both in the short term (60 min) and in the long term (7-28 d) as compared with A. stolonifera, when temperatures increased from 17 degrees C to 27 degrees C or from 27 degrees C to 37 degrees C. Thermal A. scabra also maintained a significantly lower maintenance cost than A. stolonifera as temperatures increased to 27 degrees C or 37 degrees C. The results suggested that root thermotolerance of thermal A. scabra was associated with both short-term and long-term respiratory acclimation to changes in temperatures. The superior ability of adjusting the rate of root respiration to compensate for increases in carbon demand during short- or long-term temperature increases in the heat-tolerant A. scabra may result in the reduction in carbon expenditure or costs for maintenance, leading to extended root survivability in high temperature soils.     

Isolation and Identification of Submergence-induced Genes in Maize Seedlings by Suppression Subtractive Hybridization

To investigate the expression profile of maize genes induced by submergence, a subtracted cDNA library of maize seedling roots was constructed using suppression subtractive hybridization (SSH). The cDNA of maize seedling roots treated with submergence (ST) was used as tester and what from untreated roots (UT) as driver. Products of the secondary PCR from the forward subtraction were cloned into T/A vector and transferred into Escherichia coli strain JM10B by electroporation. Four hundred and eight randomly chosen transformants carrying cDNA fragments were screened with PCR-Select Deferential Screening Kit. One hundred and eighty-four cDNA clones were identified as submergence specifically induced or highly expressed. After sequencing and removing redundant cDNAs, we got 95 submergence-induced cDNA clones. Of the 95 cDNA clones, 68 contain the regions with 60%-90% identity to their homolog in GenBank, 21 are expected to be novel genes, only 6 correspond to the published maize sequences. Key words: maize; expression profile; suppression subtractive hybridization (SSH); submergence