Expression analysis of proline metabolism-related genes from halophyte Arabis stelleri under osmotic stress conditions

Arabis stelleri var.japonica evidenced stronger osmotic stress tolerance than Arabidopsis thaliana.Using an A.thaliana microarray chip,we determined changes in the expression of approximately 2 800genes between A.stelleri plants treated with 0.2 M mannitol versus mock-treated plants.The most significant changes in the gene expression patterns were in genes defining cellular components or in genes associated with the endomembrane system,stimulus response,stress response,chemical stimulus response,and defense response.The expression patterns of three de novo proline biosynthesis enzymes were evaluated in A.stelleri var.japonica seedlings treated with 0.2 M mannitol,0.2 M sorbitol,and 0.2 M NaCl.The expression of Δ1-pyrroline-5-carboxylate synthetase was not affected by NaCl stress but was similarly induced by mannitol and sorbitol.The proline dehydrogenase gene,which is known to be repressed by dehydration stress and induced by free L-proline,was induced at an early stage by mannitol treatment,but the level of proline dehydrogenase was increased later by treatment with both mannitol and NaCl.The level of free L-proline accumulation increased progressively in response to treatments with mannitol,sorbitol,and NaCl.Mannitol induced L-proline accumulation more rapidly than NaCl or sorbitol.These findings demonstrate that the osmotic tolerance of the novel halophyte,Arabis stelleri,is associated with the accumulation of L-proline.     

Expression Stabilities of Candidate Reference Genes for RT-qPCR under Different Stress Conditions in Soybean

Due to its accuracy, sensitivity and high throughput, real time quantitative PCR (RT-qPCR) has been widely used in analysing gene expression. The quality of data from such analyses is affected by the quality of reference genes used. Expression stabilities for nine candidate reference genes widely used in soybean were evaluated under different stresses in this study. Our results showed that EF1A and ACT11 were the best under salinity stress, TUB4, TUA5 and EF1A were the best under drought stress, ACT11 and UKN2 were the best under dark treatment, and EF1B and UKN2 were the best under virus infection. EF1B and UKN2 were the top two genes which can be reliably used in all of the stress conditions assessed.     

超表达AhNCED1拟南芥植株在渗透胁迫下抗氧化能力和抗旱相关下游基因表达变化

利用纯系超表达AhNCED1拟南芥植株,在添加300mmol·L-1山梨醇的MS培养基上胁迫培养,分析其表型及抗氧化能力。结果表明,在正常情况及300mmol·L-1山梨醇处理10d条件下,与129B08/nced3突变体和野生型比较,超表达AhNCED1植株生长状况好,主根长度增加,幼苗体内O2ˉ含量水平较低,POD和SOD酶活性较高。说明NCED1基因对于拟南芥体内抗氧化酶系统的维持有重要意义。用sqRT-PCR方法检测结果表明,野生型和超表达AhNCED1拟南芥植物体内KIN1、COR47和RAB18基因表达随脱水延长而明显增强,且在超表达AhNCED1拟南芥中增强幅度大。     

Compatible-Solute-Supported Periplasmic Expression of Functional Recombinant Proteins under Stress Conditions

The standard method of producing recombinant proteins such as immunotoxins (rITs) in large quantities is to transform gram-negative bacteria and subsequently recover the desired protein from inclusion bodies by intensive de- and renaturing procedures. The major disadvantage of this technique is the low yield of active protein. Here we report the development of a novel strategy for the expression of functional rIT directed to the periplasmic space of Escherichia coli. rITs were recovered by freeze-thawing of pellets from shaking cultures of bacteria grown under osmotic stress (4% NaCl plus 0.5 M sorbitol) in the presence of compatible solutes. Compatible solutes, such as glycine betaine and hydroxyectoine, are low-molecular-weight osmolytes that occur naturally in halophilic bacteria and are known to protect proteins at high salt concentrations. Adding 10 mM glycine betaine for the cultivation of E. coli under osmotic stress not only allowed the bacteria to grow under these otherwise inhibitory conditions but also produced a periplasmic microenvironment for the generation of high concentrations of correctly folded rITs. Protein purified by combinations of metal ion affinity and size exclusion chromatography was substantially stabilized in the presence of 1 M hydroxyecotine after several rounds of freeze-thawing, even at very low protein concentrations. The binding properties and cytotoxic potency of the rITs were confirmed by competitive experiments. This novel compatible-solute-guided expression and purification strategy might also be applicable for high-yield periplasmic production of recombinant proteins in different expression systems.     

东乡野生稻及其渐渗系应答低磷胁迫的基因差异表达分析

以东乡野生稻耐低磷渐渗系IL171及其双亲(栽培稻‘协青早B’和东乡野生稻)为试材,采用cDNA-AFLP技术分析其幼苗期应答低磷胁迫的差异表达谱特征,并采用实时荧光定量PCR分析验证差异表达基因的表达特性,为探究东乡野生稻耐低磷胁迫的分子机制、发掘耐低磷相关的基因奠定基础。结果显示:(1)基于17对扩增效果较好的cDNA-AFLP引物分析发现,不同胁迫时间的参试材料与其对照组(正常磷水平)相比,具有很多(20~159个)上调或下调表达的差异片段。(2)与‘协青早B’相比,IL171中特异性上调的差异带有36条,下调表达61条,而东乡野生稻中分别有79条和136条;IL171与东乡野生稻共有的上调差异带为13条,下调差异带有15条。(3)回收纯化其中60条特异性差异表达条带,最终克隆测序获得50个差异表达基因片段TDFs;通过Blast比对和功能分析,可将TDFs分为8类,包括能量与代谢、基因表达调控、信号转导和转录因子等。(4)实时荧光定量PCR验证其中6个TDFs发现,各差异片段的荧光定量PCR表达模式与cDNA-AFLP结果一致,表明该实验的cDNAAFLP差异表达结果可靠,东乡野生稻的部分耐低磷相关基因已成功导入到渐渗系中,是发掘利用东乡野生稻耐低磷相关基因、探究其耐低磷分子机制的重要资源。     

外源茉莉酸甲酯对盐胁迫下小桐子幼苗渗透调节和脯氨酸代谢的影响

以小桐子幼苗为材料,采用人工气候箱内水培试验,设置不同浓度(0、20、40、60、80、100μmol·L^(-1))茉莉酸甲酯(MeJA)和150 mmol·L^(-1)NaCl胁迫处理,分析不同处理条件下小桐子幼苗叶片的组织活力、MDA含量、水势、含水量和叶片渗透调节物质脯氨酸、甜菜碱和可溶性糖的含量,以及脯氨酸代谢关键酶P5CS、OAT和甜菜碱合成关键酶BADH活性和相关基因表达水平,探讨外源茉莉酸甲酯对盐胁迫下小桐子幼苗渗透调节能力的影响及其机制。结果表明:(1)外源MeJA处理可提高盐胁迫下小桐子幼苗叶片的组织活力和叶片含水量,降低小桐子幼苗叶片的MDA含量和水势,且60μmol·L^(-1)浓度处理效果最佳。(2)外源MeJA处理可提高盐胁迫下小桐子幼苗叶片的脯氨酸、甜菜碱和可溶性糖的含量,且60μmol·L^(-1)MeJA处理显著提高了盐胁迫下小桐子幼苗内源茉莉酸、脯氨酸和甜菜碱的含量。(3)60μmol·L^(-1)MeJA也提高了盐胁迫下小桐子BADH、P5CS和OAT的活性,并上调了JcBADH、JcP5CS、JcOAT基因的表达水平,但MeJA降低了脯氨酸降解酶ProDH的活性,下调了JcProDH基因的表达。研究发现,在盐胁迫条件下,外源MeJA通过活化脯氨酸生物合成的谷氨酸和鸟氨酸途径,尤其是鸟氨酸途径,以及抑制脯氨酸降解途径来促进小桐子幼苗脯氨酸的积累,同时MeJA也激活了幼苗体内甜菜碱的生物合成过程,从而强化了盐胁迫下幼苗的渗透调节作用和耐盐性,表明MeJA诱导的渗透调节在小桐子耐盐性形成过程中发挥着重要作用。     

Over-expression of Osmotin Induces Proline Accumulation and Confers Tolerance to Osmotic Stress in Transgenic Tobacco

Osmotin has been implicated in conferring tolerance to drought and salt stress in plants. We have over-expressed the osmotin gene under the control of constitutive CaMV 35S promoter in transgenic tobacco, and studied involvement of the protein in imparting tolerance to salinity and drought stress. The transgenic plants exhibited retarded leaf senescence and improved germination on a medium containing 200mM NaCl. Further, the transgenics maintained higher leaf relative water content (RWC), leaf photosynthesis and free proline content than the wild type plants during water stress and after recovery from stress. When subjected to salt stress (200mM NaCl), the transgenic plants accumulated significantly more proline than the wild type plants. These results suggest the involvement of the osmotin-induced increase in proline in imparting tolerance to salinity and drought stress in transgenic plants over-expressing the osmotin gene.     

盐胁迫条件下玉米萜类合成相关基因的表达分析

萜类是植物生长发育中具有重要作用的次级代谢产物,某些萜类可被生物和非生物胁迫诱导产生。用200 mmol/L NaCl溶液处理商用玉米品种天塔五号（F1）及其父母本三叶期幼苗,qRT-PCR结果发现萜类合成途径中的萜烯合酶基因2（terpene synthase 2, TPS2）、萜烯合酶基因3（terpene synthase 3, TPS3）、牻牛儿基牻牛儿基焦磷酸合成酶基因4（geranylgeranyl diphosphate synthase 4, GGPS4）在三组材料中表达量随胁迫时间延长均先上调再下调,且F1表达量显著高于亲本。F1自交F2代耐盐性及基因表达情况分析表明,F2代中三个基因表达仍与耐盐性呈正相关。对盐胁迫条件下总类胡萝卜素及各成分含量、生物量、光合指标、叶绿素和脯氨酸含量进行测定,发现F1耐盐能力明显优于亲本,表明 TPS2、TPS3、GGPS4 基因高水平表达与F1发挥耐盐优势、抵御逆境胁迫有一定相关性。     

水曲柳S6K基因的生物信息学及胁迫和激素下的表达分析

以所获取的水曲柳FmS6K基因（FmS6K1和FmS6K2）为对象,解析其核苷酸序列及其所编码蛋白质的基本信息,探索非生物胁迫和植物激素条件下基因的表达特征。结果表明,FmS6K1和FmS6K2基因长度分别为1 583和1 796 bp,分别编码480和483个氨基酸,二者均含有完整的开放阅读框。FmS6K1为两性蛋白,FmS6K2是亲水蛋白,二者均不含有信号肽。低温（4℃）和盐（NaCl）胁迫均影响FmS6K1和FmS6K2基因的表达,水曲柳根、茎、叶中的FmS6K1和FmS6K2基因对盐更加敏感,FmS6K2基因比FmS6K1基因对低温和盐胁迫的响应更为敏感;外源ABA、GA和IAA均能够影响FmS6K1和FmS6K2基因的表达,FmS6K1在水曲柳根、茎、叶中对外源的IAA响应更大;水曲柳根茎叶中FmS6K2基因均对外源ABA处理有显著上调应答,对外源GA的响应主要在根中,但对IAA的应答主要在茎和叶片中。上述结果表明FmS6K基因在调控植物生长发育和适应逆境胁迫方面扮演重要角色。     

Expression of Plasma Membrane Water Channel Genes under Water Stress in Nicotiana excelsior

Deduced amino acid sequences encoded by the cDNAs related tothe MIP gene family from Nicotiana excelsior were characterized.Phylogenetic characterization of the products of correspondinggenes named NeMip1, NeMip2, and NeMip3 strongly suggested thatthey are water channel proteins localized in the plasma membrane.Organ specificity of the gene expression was examined in leaves,roots, and reproductive organs. NeMip1 was expressed in rootsand reproductive organs; however, it was hardly detectable inleaves. Two other genes, NeMip2 and NeMip3, were expressed inall of organs examined. mRNA accumulation from the genes wasinvestigated in leaves under salt- and drought-stresses. Theresults demonstrated that mRNA accumulation from all three genesincreased under salt- and drought-stresses within one day. However,they showed different accumulation patterns. In addition totheir up-reg-ulation under salt- and drought-stresses, dailychanges in NeMip2 and NeMip3 mRNA accumulation was observedunder unstressed conditions in leaves. (Received May 2, 1997; Accepted September 3, 1997)     

NaCl胁迫下宁夏枸杞光合作用相关基因差异表达分析

宁夏枸杞是著名的耐盐药用植物,该研究通过室内水培试验,利用高通量测序技术和qRT-PCR技术检测了不同浓度NaCl胁迫(0、100、200、300 mmol/L)下宁夏枸杞叶片光合作用相关基因差异表达,并分析了其叶绿素含量、净光合速率、Rubisco活性的变化,以揭示宁夏枸杞在盐胁迫条件下光合机构及光合作用相关基因差异表达规律,为深入解析宁夏枸杞响应盐胁迫的光合机理奠定基础。结果表明：(1)用100、200、300 mmol/L NaCl胁迫处理7 d时宁夏枸杞分别有14、26、55个光合作用相关基因差异表达,且随着NaCl胁迫程度的增加下调表达基因多于上调表达基因。(2)qRT-PCR结果显示,宁夏枸杞的3个光合作用相关基因ATPε、CLH2、Lhcb3的相对表达量均随着NaCl胁迫程度的加深总体呈显著下降的趋势,qRT-PCR验证结果与RNA-seq测序结果基本一致。(3)随着NaCl胁迫程度的增加,宁夏枸杞叶片中的叶绿素a和叶绿素b含量以及净光合速率和Rubisco活性均呈显著下降的趋势,而类胡萝卜素含量变化不显著。研究认为,宁夏枸杞能通过诱导光合作用相关基因的差异表达来调控叶片...     

Light-Dark Changes in Proline Content of Barley Leaves under Salt Stress

Proline accumulation in leaves of barley (Hordeum vulgare L. cv. Alfa) seedlings treated with 150 mM NaCl was promoted in the light and suppressed in the dark. The light/dark changes of proline content was enhanced with each 12 h light/12 h dark cycle and the proline content increased steadily. Root and shoot concentrations of Na⁺ and Cl^– in salt treated plants increased about 10 to 25 times as compared to the control. The content of these ions and the content of malondialdehyde were higher in the shoot of seedlings exposed to salt stress for 4 d in the light in comparison with the seedlings exposed to NaCl for 4 d in darkness. Light stimulated both ions and proline accumulation in the leaves and has no effect in the roots. Oxygen uptake was higher in the seedlings kept 4 d in the light which have higher endogenous free proline content. Chlorophyll fluorescence measurements showed that the photochemical activity of PS 2 slightly decreased as a result of salt stress and was not influenced by light regimes during plant growth.