Na2CO3胁迫对星星草叶肉细胞超微结构的影响

利用透射电镜技术对Na2CO3胁迫下星星草叶肉细胞超微结构进行了观察。结果表明：未胁迫的叶肉细胞排列疏松,各种细胞器结构完整,叶绿体含少量淀粉粒和脂质球。轻度盐胁迫（2g/L,4g／LNa2CO3）对叶肉细胞超微结构影响较小。中度盐胁迫（6g／L,8g／L Na2CO3）引起叶肉细胞超微结构的变化,叶绿体类囊体肿胀,基粒紊乱,不含淀粉粒,脂质球数量增加,叶绿体由原来的梭形或椭球形变成圆球状;部分线粒体嵴消失,出现晶体结构;中央大液泡破裂;核逐渐降解。高度盐胁迫（10g／L,12g／LNa2CO3）下,叶绿体片层结构消失,脂质球数量增加,体积变大,被大量的膜片层所包围,叶绿体内、外膜消失,叶肉细胞中看不到叶绿体的存在;膜片层包围线粒体;叶肉细胞中可见大量的泡状结构和膜片层,叶肉细胞死亡。上述结果表明,细胞器特别是叶绿体膜结构的破坏与盐胁迫叶肉细胞最终死亡密切相关。     

Na2 CO3胁迫下星星草幼苗活性氧及保护酶活性的变化

对低浓度Na2CO3胁迫下星星草幼苗相对电导率、O2.—产生速率、H2O2含量以及保护酶CAT、SOD和POD活性的研究结果表明,低盐胁迫1 d后,星星草幼苗细胞膜的通透性、O2.—产生速率、H2O2含量及保护酶活性都随着盐胁迫的加剧而升高,其具体的变化规律与盐胁迫强度和幼苗细胞膜的受损伤程度密切相关,但相关关系的性质上具有差异。     

星星草耐盐碱生理机制再探讨

用不同浓度Na2CO3处理星星草幼苗,7d后测定叶片相对电导率、渗透势、光合速率（Pn）、气孔导度（Gs）、胞间CO2浓度（Ci）、蒸腾强度（Tr）、水分利用效率（WUE）等生理指标,以研究星星草抗盐碱的生理机制。结果表明,低浓度Na2CO3胁迫下,星星草幼苗可通过渗透调节来减轻细胞所受的伤害,因而膜透性受影响较小,相对电导率上升较少,而Gs基本保持不变,Pn、Tr还有所促进,WUE有所增加,这些结果说明,星星草幼苗对低浓度碱性盐具有一定抗性;但犀星草幼苗的这种抗性有一定限度,随着Na2CO3胁迫浓度的增加,Gs、Tr、Pn依次受到抑制,最终膜透性急剧增加,说明星星草幼苗已受损严重,这些变化具有较强的顺序性。     

Na2CO3胁迫下星星草(Puccinellia tenuiflora)幼苗叶表皮和叶肉细胞中K、Na的相对含量

对不同强度Na2CO3胁迫处理下星星草幼苗叶片表皮和叶肉细胞中K、Na的透射电镜X-射线电子探针显微分析和叶片表面扫描电镜X-射线电子探针显微分析,结果表明：在相同胁迫强度下,无论是表皮细胞还是叶肉细胞的细胞壁和液泡中的Na相对含量均明显高于细胞质中的Na相对含量,并且K的相对含量均明显比相应部位Na的相对含量高,细胞壁与液泡中的Na相对含量变化范围非常接近。在Na2CO3胁迫浓度低于0．1molL^-1时,在相同胁迫强度下,K的相对含量高于Na的相对含量,使细胞质保持相对高的K／Na比。而尽管向细胞壁和液泡分流了大量的Na,但是细胞质中的Na相对含量仍然随着Na2CO,胁迫强度的增加而增加,一方面证明星星草在Na2CO3胁迫下维持相对高的K／Na比的能力是有一定限度的,另一方面暗示星星草作为盐生植物在盐碱环境中一定程度上Na可以部分地代替K而行使部分K的生理功能。     

Na2CO3胁迫下星星草幼苗叶绿体GST活性变化及其与相关指标的关系

通过对不同浓度Na2CO3胁迫下星星草幼苗培养基质和叶片渗透势、叶片相对电导率、叶绿体谷胱甘肽转移酶（GST）活性和O2^-产生速率的研究,以探讨星星草幼苗叶绿体GST活性在Na2CO3胁迫下的变化规律及其在抗Na2CO3胁迫中的作用。结果表明,在0～0．4％Na2CO3胁迫范围内,随着其浓度的增加星星草幼苗叶绿体GST活性增强,Na2CO3胁迫浓度继续增加则GST活性急剧减弱。星星草幼苗叶绿体GST活性随培养基质渗透势、叶片渗透势和叶片相对电导率以及叶绿体O2产生速率的变化趋势相似。叶绿体GST活性和Na2CO3胁迫浓度以及叶片渗透势之间、叶绿体GST活性和O2^-产生速率以及叶片渗透势之间、叶绿体GST活性和培养基质渗透势以及叶片渗透势之间、叶绿体GST活性和O2^-产生速率以及叶片相对电导率之间相关关系显著。说明叶绿体GST在星星草幼苗抵抗低强度（浓度）Na2CO3胁迫中起着非常重要的作用。     

Physiological and proteomic analysis of salinity tolerance in Puccinellia tenuiflora

Soil salinity poses a serious threat to agriculture productivity throughout the world. Studying mechanisms of salinity tolerance in halophytic plants will provide valuable information for engineering plants for enhanced salt tolerance. Monocotyledonous Puccinellia tenuiflora is a halophytic species that widely distributed in the saline-alkali soil of the Songnen plain in northeastern China. Here we investigate the molecular mechanisms underlying moderate salt tolerance of P. tenuiflora using a combined physiological and proteomic approach. The changes in biomass, inorganic ion content, osmolytes, photosynthesis, defense-related enzyme activities, and metabolites in the course of salt treatment were analyzed in the leaves. Comparative proteomic analysis revealed 107 identities (representing 93 unique proteins) differentially expressed in P. tenuiflora leaves under saline conditions. These proteins were mainly involved in photosynthesis, stress and defense, carbohydrate and energy metabolism, protein metabolism, signaling, membrane, and transport. Our results showed that reduction of photosynthesis under salt treatment was attributed to the down-regulation of the light-harvesting complex (LHC) and Calvin cycle enzymes. Selective uptake of inorganic ions, high K(+)/Na(+) ratio, Ca(2+) concentration changes, and an accumulation of osmolytes contributed to ion balance and osmotic adjustment in leaf cells. Importantly, P. tenuiflora plants developed diverse reactive oxygen species (ROS) scavenging mechanisms in their leaves to cope with moderate salinity, including enhancement of the photorespiration pathway and thermal dissipation, synthesis of the low-molecular-weight antioxidant α-tocopherol, and an accumulation of compatible solutes. This study provides important information toward improving salt tolerance of cereals.     

Difference Between Salt(NaCl)and Alkaline(Na2CO3)Stresses on Puccinellia tenuiflora(Griseb. )Scribn. et Merr. Plants

Eight-week-old plants of Puccinellia tenuiflora (Griseb.) Scribn. et Merr. growing in pots filled with vermiculite were stressed by treating with 12.5–800 mmol/L solutions of neutral satt(NaC1) or basic salt(Na2CO3). Strain indexes such as relative growth rate etc. were determined. There was a significant difference between the two kinds of stresses. Maximum stress value that P. tenuiflora plants can tolerate is 60 mmol/L for the neutral salt and 200 mmol/L for the basic salt Na2CO3. Under NaCl-stress, great amount of proline accumulated, and citric acid content gradually decreased. But under Na2CO3-stress, proline content did not raise too much and citric acid content obviously increased with the increasing stress value. Under both stress conditions, Na+ content increased and K+ content decreased with the increasing stress value, but the effect of NaCl-stress on K+ content in roots and shoots was much less than that of Na2CO3-stress. In both stresses, the elevation of electrolyte leakage rate of leaf orchestrated with the change of stress value. This finding represented the only similarity among the strain indexes determined in both stresses.     

Effects of Na2CO3 and NaCl stresses on the antioxidant enzymes of chloroplasts and chlorophyll fluorescence parameters of leaves of Puccinellia tenuiflora (Turcz.) scribn.et Merr.

Antioxidant enzymes in chloroplasts and chlorophyll fluorescence parameters of leaves of Puccinellia tenuiflora (Turcz.) scribn.et Merr. under isotonic Na₂CO₃ and NaCl stresses were studied. Ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities showed a similar increasing trend and then decreased with the decreasing osmotic potential of culture solution, peaking at −4.74 × 10⁵ Pa under NaCl stress and at −3.40 × 10⁵ Pa under Na₂CO₃ stress. APX, glutathione transferase and SOD activities were higher under NaCl stress than those under Na₂CO₃ stress, and higher activities of antioxidant enzymes in chloroplasts were accompanied by lower MDA content under NaCl stress. F _v/F _m, F _v/F _o and F _v′/F _m′ all initially increased and then decreased with the decreasing osmotic potential of culture solution, while Φ _PSII, qNP and HDR showed a constant increase. F _v/F _m, F _v/F _o, Φ _PSII and qNP under NaCl stress were also shown to be higher than those under Na₂CO₃ stress. The present study suggested that acidity played an important role in the hurt toPuccinellia tenuiflora seedlings, which was due to higher activities of antioxidant enzymes, qNP and Φ _PSII, and the Na₂CO₃ resistance to Puccinellia tenuiflora was also supposed to be less than NaCl resistance in present work.     

过量表达星星草PtSOS_1提高拟南芥的耐盐性

将星星草中分离的质膜型Na+/H+逆向转运蛋白基因PtSOS1(GenBank登录号EF440291)构建到pGWB2植物表达载体上,转化拟南芥,获得抗卡那霉素的抗性植株.PCR和Northern检测表明,PtSOS1已整合到拟南芥基因组中并过量表达.耐盐性实验表明,PtSOS1过量表达提高了拟南芥植株的耐盐性.盐分测定表明,盐胁迫下PtSOS1转基因植株中Na+积累低于野生型的,K+含量则高于野生型的,转基因植株中K+/Na+比值高于野生型.     

朝鲜碱茅抗坏血酸过氧化物酶的基因克隆和表达

构建到酵母表达载体pYES2-PutAPx,并导入酵母IVSC1菌株后在半乳糖的诱导下分析具有抗氧化性.本研究成功克隆了抗坏血酸过氧化物酶基因(PutA Px)编码区,并做了初步分析,为进一步研究逆境诱导的氧化胁迫的作用机理研究奠定了基础.     

拒盐型盐生植物小花碱茅肌动蛋白基因片段的克隆及序列分析

本研究根据其它植物Actin基因的保守序列设计一对简并性引物,以拒盐型盐生植物小花碱茅根部总RNA为模板,采用RT-PCR的方法扩增出Actin基因片段并克隆到PUCm-T载体,阳性克隆经PCR检测后进行测序,在GenBank中注册;序列分析结果表明:该片段长约600 bp,编码198个氨基酸;所得序列与GenBank中注册的其它植物Actin基因序列同源性均在84%以上,与其它肌动蛋白的氨基酸序列同源性达94%以上.     

Na2CO3胁迫对黄瓜幼苗生长及生理指标的影响

以‘津优一号’和‘春四’黄瓜为实验材料,采用砂培法研究了Na2CO3胁迫对黄瓜幼苗生长及生理指标的影响。结果显示,随着Na2CO3浓度的增大,两个黄瓜品种的幼苗根冠比逐渐下降,叶绿素含量先上升后下降,可溶性糖含量、脯氨酸含量、SOD活性逐渐上升;‘津优一号’的相对电导率和丙二醛含量随Na2CO3浓度的增大逐渐增加,而‘春四’的相对电导率和丙二醛含量在20 mmol.L-1时最低,此后逐渐上升。主成分分析结果显示,丙二醛含量、SOD活性、可溶性糖含量、脯氨酸含量、叶绿素b,这5项指标在评价黄瓜幼苗的Na2CO3耐性方面是比较重要的。结合这5项指标综合分析,发现‘津优一号’较‘春四’有较强的耐碱性。     

Effects of NaCl and Na2CO3 stresses on photosynthetic ability of Chlamydomonas reinhardtii

Chloride and carbonate salts are the main salts causing salinization and widely exist in aquatic environment, so algae may suffer from salinization stress for high water evaporation. In this study, in order to investigate and compare the toxic effects of the two salts on algal photosynthesis, we used NaCl and Na₂CO₃ to stress Chlamydomonas reinhardtii. Under the two salt stresses, the content of O ₂ ^?· and H₂O₂ in the cells was increased significantly, and it was much higher in Na₂CO₃ treatment than in NaCl treatment at the same Na⁺ concentration. The absorbance spectra and 4^th derivative spectra of photosynthetic pigments were declined under 300 mM NaCl and 25 mM Na₂CO₃ stresses, and remarkably changed under 50 mM and 100 mM Na₂CO₃ stresses. When the cells stressed by the two salts, the maximum quantum yield (Fv/Fm), electron transport rate (ETR) and photochemical quenching (qP) were reduced markedly, but the nonphotochemical dissipation (NPQ) was increased markedly. At the same Na⁺ concentration, Na₂CO₃ stress had stronger toxic effects on photosynthetic ability than NaCl stress.     

星星草铁蛋白基因PtFer的克隆及表达分析

目的:从星星草中克隆一个铁蛋白相关基因,分析其序列特征及基因表达模式。方法与结果:构建星星草RACE cDNA文库,根据GenBank中报道的铁蛋白基因EST序列设计引物,利用RACE方法克隆得到星星草铁蛋白基因PtFer的全长cDNA序列(GenBank登录号为HM125047);序列分析表明,PtFer的核苷酸序列长度为751 bp,开放读框为336 bp,编码111个氨基酸残基,预测蛋白质相对分子质量为12.8×103。其蛋白质序列具有铁蛋白的特征性保守区域,与水稻属同一进化分支,与其他禾本科植物铁蛋白的序列相似性达80%以上。Northern杂交分析表明,PtFer的表达量随Na2CO3的浓度和时间的增加而升高,并在12～24 h内持续保持较高的表达水平。结论:用分子生物学及生物信息学技术克隆并分析了星星草铁蛋白基因PtFer的全长cDNA序列及编码蛋白的结构,并初步探索了盐碱胁迫下其表达模式,为研究植物耐盐碱机理奠定了基础。     

NaHCO3胁迫下星星草根中Ca2+与Ca2+-ATPase 的超微细胞化学定位

利用焦锑酸盐和磷酸铅沉淀技术分别对NaHCO₃胁迫条件下星星草(Puccinellia tenuiflora)根中Ca²⁺和Ca²⁺-ATPase 进行超微细胞化学定位研究, 旨在进一步探讨Ca²⁺在NaHCO₃胁迫诱导胞内信号转导过程中的作用, 以及Ca²⁺-ATPase活性定位变化与NaHCO₃胁迫下星星草抗盐碱能力的关系。结果表明: 在正常状态下, 根毛区细胞质内Ca²⁺较少, 主要位于质膜附近和液泡中, Ca²⁺-ATPase主要定位于质膜和液泡膜, 有一定活性。在0.448%NaHCO₃胁迫下, 根毛区细胞质中Ca²⁺增多, 液泡中Ca²⁺减少, 且主要集中于液泡膜附近, 质膜和液泡膜Ca²⁺-ATPase活性明显升高。在1.054%NaHCO₃胁迫下,细胞质中分布的Ca²⁺增多, 而液泡中Ca²⁺极少, Ca²⁺-ATPase活性也降低。以上结果表明, Ca²⁺亚细胞定位和Ca²⁺-ATPase活性变化在星星草响应NaHCO₃胁迫的信号传递过程中具有重要作用。     

NaHCO3胁迫下星星草根中Ca2＋与Ca2＋-ATPase的超微细胞化学定位

利用焦锑酸盐和磷酸铅沉淀技术分别对NaHCO3胁迫条件下星星草（Puccinellia tenuiflora）根中Ca2＋和Ca2＋-ATPase进行超微细胞化学定位研究,旨在进一步探讨Ca2＋在NaHCO3胁迫诱导胞内信号转导过程中的作用,以及Ca2＋-ATPase活性定位变化与NaHCO3胁迫下星星草抗盐碱能力的关系。结果表明：在正常状态下,根毛区细胞质内Ca2＋较少,主要位于质膜附近和液泡中,Ca2＋-ATPase主要定位于质膜和液泡膜,有一定活性。在0.448%NaHCO3胁迫下,根毛区细胞质中Ca2＋增多,液泡中Ca2＋减少,且主要集中于液泡膜附近,质膜和液泡膜Ca2＋-ATPase活性明显升高。在1.054%NaHCO3胁迫下,细胞质中分布的Ca2＋增多,而液泡中Ca2＋极少,Ca2＋-ATPase活性也降低。以上结果表明,Ca2＋亚细胞定位和Ca2＋-ATPase活性变化在星星草响应NaHCO3胁迫的信号传递过程中具有重要作用。     

星星草RAPD-PCR反应体系建立与优化

目的:寻找一个可用于星星草(Puccinellia tenuiflora(Griseb.)Scribn.et Merr)RAPD-PCR的最适宜的反应体系。方法:利用正交设计法对影响PCR扩增效果的一些因素诸如TaqDNA聚合酶的用量、Mg2 浓度、dNTP以及引物浓度等指标进行筛选和优化。然后,通过引物对该优化结果进行验证。结果:正交设计结果表明,最适宜的PCR反应条件:20μl PCR反应体系中包括10×buffer2μl、模板DNA20ng、dNTP1.6mmol/L、TaqDNA聚合酶0.35U、引物1.0mmol/L、Mg2 1.8mmol/L。验证结果表明,该体系扩增出的条带多且清晰。结论:该研究得出的体系是适合RAPD-PCR的最适宜的反应体系,为今后星星草遗传多样性的研究奠定了基础。     

差异显示技术研究NaHCO_3胁迫下星星草基因表达

用差异显示技术研究NaHCO3胁迫下星星草(Puccinelliatenuiflora)基因的表达。经ReverseNorth-ern检测,获得了7个差异表达的基因片段。其中,6个为胁迫后诱导表达,1个为胁迫后抑制表达。序列同源性分析表明,胁迫诱导表达的6个基因片段中,1个与钙依赖性蛋白激酶(CDPKs)基因同源性较高,其余5个可能为新序列,胁迫后抑制表达的基因片段与假定的adaptor蛋白基因同源性较高。本研究为进一步研究星星草的抗盐机理奠定了基础。     

差异显示技术研究NaHCO3胁迫下星星草基因表达

用差异显示技术研究NaHCO3胁迫下星星草(Puccinellia tenuiflora)基因的表达.经Reverse Northern检测,获得了7个差异表达的基因片段.其中,6个为胁迫后诱导表达,1个为胁迫后抑制表达.序列同源性分析表明,胁迫诱导表达的6个基因片段中,1个与钙依赖性蛋白激酶(CDPKs)基因同源性较高,其余5个可能为新序列,胁迫后抑制表达的基因片段与假定的adaptor蛋白基因同源性较高.本研究为进一步研究星星草的抗盐机理奠定了基础.