在胡杨NHX基因内发现细菌转座子IS10-L的存在

采用RT-PCR扩增的方法,从新疆不同地区的野生植物胡杨中分别克隆获得lkb和2．3kb的cDNA片段,测序和序列分析表明这两个基因片段均包含NHX基因的部分读码框架,分别命名为PtNHX和PwNHX。PtNHX序列同源性分析结果显示胡杨NHX基因与滨藜NHX基因同源性高达98％,与碱蓬同源性达到86%,与拟南芥同源性为84％,与水稻同源性为80％,表明它是植物中高度保守的一种基因,同时说明野生植物胡杨中也存在与拟南芥相似的植物耐盐相关基因。PwNHX序列分析表明,该基因内部含有一种转座酶的读码框,大小约1350bp,与已发表的Shigella flexneri．转座子Th10基因序列(AF162223)同源性为99％。NHX基因的蛋白产物在植物的耐盐性方面起着重要的作用。推测PwNHX。由于插入转座酶读码框可能会导致该基因的功能丧失。胡杨生存于盐碱地,其体内可能存在另一些机制使胡杨具有抗盐碱的能力。对于这些机制的研究可能有助于进一步了解植物的抗盐机制。利用PwNHX基因内的转座子作基因标签,可进一步研究胡杨的其它基因,从而有可能揭示胡杨叶子发育的变态过程、耐盐碱等性状．     

过表达胡杨XTH基因能够提高烟草抗旱性

胡杨是典型的抗旱树种。挖掘和鉴定胡杨的耐旱基因对于提高植物抗旱性具有重要意义。木葡聚糖内转糖苷酶/水解酶（XTH）是植物细胞壁重构过程中的关键酶,在植物逆境胁迫响应中发挥重要作用。我们前期已从胡杨叶片中克隆了PeXTH基因。本文利用Real-time PCR检测PeXTH基因在干旱胁迫下的表达水平。在此基础上,构建植物表达载体pMDC85-PeXTH,通过农杆菌介导法将PeXTH基因转入烟草,分析过表达PeXTH基因烟草的抗旱性。研究发现,胡杨叶片中PeXTH基因的表达受干旱胁迫诱导。干旱处理后,转PeXTH基因烟草的萌发率明显高于野生型烟草;与野生型植株相比,转基因植株的叶片失水速率明显降低。干旱胁迫下,转基因烟草的气孔开度仅为野生型烟草的51.2%～53.6%。结果表明,过表达PeXTH基因能够提高烟草的抗旱性。本研究丰富了对胡杨PeXTH基因功能的认识,为植物抗旱分子育种提供了重要的基因资源。     

胡杨细胞和组织结构与其耐盐性关系的研究

The characteristics of the cell and organ structures of Populus euphratica Oliv. in relation to salt and osmotic tolerance were compared with those of P. tomentosa Cart. in vitro under the electron and light microscopic observation. P. euphratica exhibited characteristic structure which was associated with salt stress. It had well-developed epidermis and exodermis in the root tip and poorly developed conducting tissue in leaf. Root hairs were formed closer to the root tips. AsP. euphratica were stressed with salt and PEG, more abundance of chondriosomes and plastids in the cytoplasm and more containing substance in the plastid were observed and the osmophilic substance was obviously displayed in the cytoplasm and in the posterior margin of the vacuole. The filamentous structure, bigger nucleus and nucleolus were visualized in the stressed suspension-cultured cells of P. euphratica. The meristemic cells in the root tip of P. euphratica could maintain their structure when the plant was subjected to 8 g/L NaCl stress. It was also demonstrated that the cell wall and plasmalemma of P. euphratica were tightly combined as a dentate form, explaining why the cell could endure severe salt or osmotic stress and resist to plasmolysis indicating that P. euphratica possesses a solid structure base as a defense to salt stress.     

胡杨无性系幼苗响应盐胁迫的miRNA表达差异研究

植物miRNA在调控基因表达、细胞周期、生物体发育、抗逆等方面起重要作用。为研究胡杨(Populus euphratica Oliv.)的耐盐机制,以1年生胡杨无性系幼苗为材料,构建具有空间代表性的盐胁迫胡杨cDNA文库,利用二代测序技术测定NaCl胁迫下和正常培养条件下胡杨叶和根miRNA表达情况。结果表明,不同的miRNA之间表达量存在明显差异,表达丰度最高的miRNA有miR156、miR157、miR165、miR166和miR167等,合计占总表达量的90%以上。胡杨根部存在特异表达的miRNA,在整个耐盐调控机制中发挥着生理调节、分子调控和信号传导等极为重要的作用。盐处理样品中发现大量响应盐胁迫的miRNA,对这些转录因子进行靶基因预测和注释后,发现很多盐胁迫响应的miRNA与NAC和SPL等重要转录因子家族相关,与前人的结论一致,另外还发现许多miRNA的调控对象是ATP酶和激素响应因子。     

SRWD: a novel WD40 protein subfamily regulated by salt stress in rice (OryzasativaL.)

By analysis with microarray data, we found that a gene encoding a novel protein containing five WD40 repeats, was regulated by salt stress in rice and named as SRWD1 (Salt responsive WD40 protein 1). By database searching, additional four SRWD1-like genes (SRWD2-SRWD5) were found in rice genome, and these five SRWD genes formed a novel WD40 subfamily. Phylogenetic analysis showed that plant SRWD proteins divided into four groups. The significant functional divergences during SRWD evolution were found. The tissue-specific and salt responsive expression profiling for SRWD genes was investigated based on microarray data. It was found that all five SRWD genes in rice were regulated by salt stress. Further, we found that SRWD1 was regulated with different patterns by salt stress in two rice cultivars responding differently to salt stress. Our study correlates WD40 proteins with salt stress in plants and provides fundamental information for the further investigation of plant SRWD proteins.     

A novel rice C2H2-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance

A cDNA for the gene ZFP182, encoding a C2H2-type zinc finger protein, was cloned from rice by RT-PCR. ZFP182 codes an 18.2 kDa protein with two C2H2-type zinc finger motifs, one nuclear localization signal and one Leu-rich domain. The DLN-box/EAR-motif, which exists in most of plant C2H2-type zinc finger proteins, does not exist in ZFP182. The expression analysis showed that ZFP182 gene was constitutively expressed in leaves, culms, roots and spikes at the adult rice plants, and markedly induced in the seedlings by cold (4 degrees C), 150 mM NaCl and 0.1 mM ABA treatments. The approximate 1.4 kb promoter region of ZFP182 gene was fused into GUS reporter gene and transformed into tobacco. The histochemical analysis revealed that GUS expression could not be detected in transformed tobacco seedlings under normal conditions, but strongly observed in tobacco leaf discs and the vascular tissue of roots treated with NaCl or KCl. Expression of ZFP182 in transgenic tobacco and overexpression in rice increased plant tolerance to salt stress. These results demonstrated that ZFP182 might be involved in plant responses to salt stress.     

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。     

胡杨木质部水分传导对盐胁迫的响应与适应北大核心CSCD

解析植物木质部导水率对逆境的响应和适应对促进植物抗逆性机理研究和受损植被恢复具有重要意义。该文以荒漠河岸林建群种胡杨(Populus euphratica)为研究对象,系统分析了胡杨幼株根、茎、叶水分传输通道对不同浓度盐胁迫的响应和适应。结果表明:(1)胡杨幼株根系对盐胁迫的敏感性高于茎和叶,盐胁迫下根系生长和根尖数显著受到抑制,根木质部易于发生栓塞,导水率明显降低。(2)胡杨幼株茎木质部导水率对盐胁迫的响应依盐浓度而定,轻度(0.05 mol·L–1 Na Cl)和中度(0.15 mol·L–1 Na Cl)盐胁迫下,胡杨可以通过协调导管输水的有效性和安全性来调节木质部的导水率,维持植物正常生长;重度(0.30 mol·L–1 Na Cl)盐胁迫下,胡杨茎木质部导管输水有效性和安全性均明显降低,木质部导水率显著下降,并伴随叶片气孔导度的显著降低,从而严重抑制了胡杨的光合和生长。     

Expression of a calcineurin gene improves salt stress tolerance in transgenic rice

Calcineurin is a Ca²⁺- and calmodulin-dependent serine/threonine phosphatase and has multiple functions in animal cells including regulating ionic homeostasis. We generated transgenic rice plants that not only expressed a truncated form of the catalytic subunit of mouse calcineurin, but also were able to grow and fertilize normally in the field. Notably, the expression of the mouse calcineurin gene in rice resulted in its higher salt stress tolerance than the non-transgenic rice. Physiological studies have indicated that the root growth of transgenic plants was less inhibited than the shoot growth, and that less Na⁺ was accumulated in the roots of transgenic plants after a prolonged period of salt stress. These findings imply that the heterologous calcineurin plays a significant role in maintaining ionic homeostasis and the integrity of plant roots when exposed to salt. In addition, the calcineurin gene expression in the stems of transgenic plants correlated with the increased expression of the Rab16A gene that encodes a group 2-type late-embryogenesis-abundant (LEA) protein. Altogether our findings provide the first genetic and physiological evidence that expression of the mouse calcineurin protein functionally improves the salt stress tolerance of rice partly by limiting Na⁺ accumulation in the roots.     

Involvement of hydrogen peroxide and nitric oxide in salt resistance in the calluses from Populus euphratica

Nitric oxide (NO) and hydrogen peroxide (H2O2) function as signalling molecules in plants under abiotic and biotic stresses. Calluses from Populus euphratica, which show salt tolerance, were used to study the interaction of NO and H2O2 in plant adaptation to salt resistance. The nitric oxide synthase (NOS) activity was identified in the calluses, and this activity was induced under 150 mM NaCl treatment. Under 150 mM NaCl treatment, the sodium (Na) percentage decreased, but the potassium (K) percentage and the K/Na ratio increased in P. euphratica calluses. Application of glucose/glucose oxidase (G/GO, a H2O2 donor) and sodium nitroprusside (SNP, a NO donor) revealed that both H2O2 and NO resulted in increased K/Na ratio in a concentration-dependent manner. Diphenylene iodonium (DPI, an NADPH oxidase inhibitor) counteracted H2O2 and NO effect by increasing the Na percentage, decreasing the K percentage and K/Na ratio. NG-monomethyl-L-Arg monoacetate (NMMA, an NO synthase inhibitor) and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (PTIO, a specific NO scavenger) only reversed NO effect, but did not block H2O2 effect. The increased activity of plasma membrane (PM) H+ -ATPase caused by salt stress was reversed by treatment with DPI and NMMA. Exogenous H2O2 increased the activity of PM H+ -ATPase, but the effect could not be diminished by NMMA and PTIO. The NO-induced increase of PM H+ -ATPase can be reversed by NMMA and PTIO, but not by DPI. Western blot analysis demonstrated that NO and H2O2 stimulated the expression of PM H+ -ATPase in P. euphratica calluses. These results indicate that NO and H2O2 served as intermediate molecules in inducing salt resistance in the calluses from P. euphratica under slat stress by increasing the K/Na ratio, which was dependent on the increased PM H+ -ATPase activity.     

互花米草NHX2基因的克隆与功能鉴定

NHX2属于CPA1基因家族,编码Na~+/H~+逆向转运蛋白,控制液泡膜中活性K~+的摄取,同时调节气孔的关闭。该研究以耐盐植物互花米草为材料,采用PCR技术克隆NHX2基因,并将其转入拟南芥进行相关功能鉴定。结果显示:(1)成功克隆获得互花米草NHX2基因CDS序列(1 602 bp),命名为SaNHX2,该基因编码533个氨基酸,SaNHX2蛋白的分子量约为58.65 kD,定位于细胞核和细胞膜,表明SaNHX2基因可能发挥转录调控的功能。(2) qRT-PCR结果显示,在ABA、NaCl和干旱胁迫处理下,互花米草叶和根中SaNHX2基因的表达量均上调。(3)为进一步鉴定其功能,成功构建植物表达载体,将SaNHX2基因转入拟南芥;经RT-PCR检测结果显示,SaNHX2基因在转基因植株中过表达;高盐胁迫处理后,转SaNHX2基因拟南芥的主根长度、叶绿素总量和相关胁迫应答基因表达量均高于转空载拟南芥,表明转SaNHX2基因拟南芥的耐盐能力显著增强。研究表明,SaNHX2基因可能在盐胁迫调节机制中发挥调控作用,可作为改良农作物耐盐的重要候选基因。     

盐生植物海滨锦葵幼苗盐胁迫下基因差异表达分析

利用cDNA-AFLP技术对海滨锦葵幼苗盐胁迫下叶片和根部的基因差异表达模式进行分析和比较, 并对部分盐胁迫应答的转录衍生片段进行了回收、测序和功能推测, 以从转录水平分析海滨锦葵的耐盐分子机制。结果显示：(1) 盐胁迫下海滨锦葵幼苗叶片和根部的基因差异表达多以量的变化为主, 包括盐胁迫下基因表达上调、下调或随盐处理浓度高低和胁迫时间长短而波动的差异表达模式; 只有少量基因的差异表达表现出质的变化, 如盐胁迫下基因沉默或诱导表达; (2) 仅在盐胁迫处理2 h的海滨锦葵幼苗根部, 基因的差异表达以质的变化为主的类型比例略高于量的变化类型比例; (3) 盐胁迫应答基因在不同组织中上调、下调、诱导或沉默的比例随胁迫处理时段而动态变化, 在刚胁迫时基因表达的差异加剧, 而后随胁迫处理时段的延长而渐趋稳定。结果预示, 从基因表达水平探讨植物的耐盐分子机理, 尽管有一定的规律可循, 但由于不同组织对盐胁迫的应答是动态变化的过程, 海滨锦葵不同组织在盐胁迫不同阶段的基因时、空、序表达特征并没有固定的程式。对部分盐胁迫下上调或诱导表达的转录衍生片段(Trivially distributed file system, TDFs)进行的序列分析和功能推测表明, 苗期海滨锦葵在盐胁迫下应答基因至少涉及3类：(1) 离子平衡重建或减少胁迫损伤相关基因(特别是运转蛋白类); (2) 恢复盐胁迫下植物生长和发育相关基因：如参与能量合成和激素调节途径相关基因等; (3)信号转导相关基因及功能未确定的新基因。文章并对盐胁迫应答基因的差异表达模式与海滨锦葵的耐盐性关系进行了讨论。     

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。     

ptc-miR801人工microRNA表达载体构建及功能初步研究

在构建盐胁迫下青杨microRNA文库中发现了ptc-miR801,为探索植物在盐胁迫条件下ptc-miR801参与胁迫应答的机制,本实验构建了植物表达载体pCAM2300-ami801,经根癌农杆菌EHA105介导、花序侵染法获得拟南芥转基因植株。RT-PCR半定量结果显示ptc-miR801可以在转基因拟南芥中超表达且NaCl胁迫下ptc-miRNA801转基因植株种子萌发率和根长显著高于野生型,说明ptc-miR801超表达增强了转基因拟南芥耐盐性。该试验为进一步研究miR801在杨树胁迫应答机制中的作用奠定基础。     

胡杨甜菜碱醛脱氢酶基因的功能分化

甜菜碱醛脱氢酶(BADH)在植物抗逆反应中发挥着重要作用。文中从胡杨cDNA克隆到2个甜菜碱醛脱氢酶基因,分别命名为PeBADH1和PeBADH2。PeBADH1和PeBADH2均编码503个氨基酸的蛋白质,预测分子量分别是54.93 kDa和54.90 kDa。组织表达模式分析发现这2个基因在正常生长、盐和H2O2胁迫下,在不同组织中的表达模式有较大差异。在大肠杆菌中表达并纯化了2个基因的重组蛋白。酶活性分析显示PeBADH1和PeBADH2蛋白对底物的活性分别是0.073μmol/(min.mg)和0.107μmol/(min.mg)。热力学稳定性分析显示这2个蛋白的热力学稳定性具有明显差异。因此,基因表达模式差异与蛋白质酶学性质的不同预示着这2个基因可能存在功能上的分化。     

CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis

Plants respond to environmental stress by activating "stress genes." The plant hormone abscisic acid (ABA) plays an important role in stress-responsive gene expression. Although Ca(2+) serves as a common second messenger in signaling stress and ABA, little is known about the molecular basis of Ca(2+) action in these pathways. Here, we show that CIPK3, a Ser/Thr protein kinase that associates with a calcineurin B-like calcium sensor, regulates ABA response during seed germination and ABA- and stress-induced gene expression in Arabidopsis. The expression of the CIPK3 gene itself is responsive to ABA and stress conditions, including cold, high salt, wounding, and drought. Disruption of CIPK3 altered the expression pattern of a number of stress gene markers in response to ABA, cold, and high salt. However, drought-induced gene expression was not altered in the cipk3 mutant plants, suggesting that CIPK3 regulates select pathways in response to abiotic stress and ABA. These results identify CIPK3 as a molecular link between stress- and ABA-induced calcium signal and gene expression in plant cells. Because the cold signaling pathway is largely independent of endogenous ABA production, CIPK3 represents a cross-talk "node" between the ABA-dependent and ABA-independent pathways in stress responses.     

小麦TaLEA1基因的表达特征及抗逆功能验证

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,因此耐盐基因挖掘对作物耐盐育种非常重要。许多研究表明胚胎发育晚期丰富蛋白(LEA)在植物应对非生物胁迫中发挥积极作用。本研究以小麦TaLEA1基因为研究对象,分析了其表达蛋白的理化性质及基因表达模式,并通过在拟南芥中过表达,分析Ta LEA1基因的抗逆功能。结果表明,TaLEA1基因的表达蛋白属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。Ta LEA1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TaLEA1基因,显著提高了盐胁迫下转基因拟南芥的种子萌发率、根长及盐和旱胁迫下的叶绿素含量。本研究结果为LEA基因抗逆机理的研究和耐盐基因的挖掘提供了重要信息。