林烟草蛋白激酶NsylCIPK3基因的克隆与功能分析

CIPK蛋白激酶家族(CBL-interacting protein kinase)是一类丝氨酸/苏氨酸蛋白激酶家族,与类钙调磷酸酶B亚基CBL(calcineurin B-like protein)蛋白共同形成CBL-CIPK网络,在植物的生长发育和逆境胁迫响应过程中发挥重要作用。烟草中该家族的研究还比较少,本研究从林烟草(Nicotiana sylvestris)中获得一个CIPK家族基因,该基因与拟南芥和杨树中的CIPK3同源性分别为68.4%和87.5%,将其命名为Nsyl CIPK3。氨基酸序列分析表明,Nsyl CIPK3具有CIPK蛋白家族的典型结构特征,在N端和C端分别具有典型的激活环结构域和NAF结构域。进化树分析显示,Nsyl CIPK3属于CIPK蛋白亚家族Ⅱ。表达模式研究表明,该基因在林烟草的叶和腋芽中的表达量相对较高,在主根中的表达量次之,在侧根、茎、花瓣和萼片中的表达量相对较低,并且在烟草成熟期的叶中表达量明显升高。在高盐、紫外光和低钾胁迫下该基因的表达发生不同程度的上调。酵母双杂交结果显示,Nsyl CIPK3可与Nsyl CBL9互作。推测Nsyl CIPK3可能通过与Nsyl CBL9互作形成信号通路,激活下游靶蛋白,参与烟草响应非生物逆境胁迫的信号转导过程。     

烟草乙烯转录因子ERF基因NtTOE3的克隆、载体构建及表达分析

ERF（Ethylene-responsive factor）是一类具有AP2特征结构域的乙烯应答转录因子,在响应植物的逆境胁迫应答中起关键作用。为明确ERF家族成员TOE3在烟草中的生物学功能和调控机制,同源克隆普通烟草K326中NtTOE3基因cDNA全长,利用实时荧光定量PCR（qRT-PCR）表征基因表达模式,结合生物信息学分析,对基因功能和调控机制进行初步预测。结果表明,该基因全长1 356 bp,编码451个氨基酸残基,预测分子量为49.84 ku。生物信息学分析表明,该蛋白是一个亲水蛋白,可能定位在细胞核,且与美花烟草（Nicotiana sylvestris）NsTOE3有96%的同源性,故命名为NtTOE3。组织表达分析发现,该基因在烟草根、茎、叶、花中均有表达,其中茎中表达量最高。逆境胁迫试验表明,该基因能快速响应低钾、高盐、干旱、H₂O₂、ABA和4℃等逆境处理。表明NtTOE3转录因子在烟草非生物胁迫中起着重要调节作用。     

花烟草NaNHX1基因的克隆及其在非生物胁迫下的表达模式

植物NHX家族基因,在植物的生长发育以及生物与非生物胁迫的应答反应中发挥着十分重要的作用。为了探究花烟草Na+/H+逆向转运蛋白的生理功能,为花烟草耐盐分子机制的研究提供参考。采用同源克隆的方法进行基因克隆,对花烟草进行非生物胁迫,并运用qPCR的方法进行基因表达模式分析。结果表明,从花烟草(Nicotiana alata)中克隆了一个属于Na+/H+逆向转运蛋白家族的基因NaNHX1。该基因的开放阅读框全长为1 599 bp,编码了532个氨基酸残基。生物信息学分析结果表明,该基因编码的蛋白分子量为58.4 kD,等电点为5.66;具有Na+/H+逆向转运蛋白家族典型的保守结构域NhaP2;该蛋白属于疏水性蛋白,包含10个跨膜区。NaNHX1基因主要定位于细胞质膜,并含有多个磷酸化位点。同源性分析的结果显示,NaNHX1基因与美花烟草(Nicotiana sylvestris)、茸毛烟草(Nicotiana tomentosiformis)以及番茄(Solanum lycoperisicum)NHX基因的亲缘关系最近,而与拟南芥的NHX基因同源性最低。NaNHX1基因的表达具有组织表达特异性,花中表达量最高,茎中次之,根和叶中表达量较低。在高盐、干旱、低温、ABA、低钾及H2O2等非生物胁迫下,NaNHX1的表达呈现3种不同的表达模式。其中,对高盐及低钾胁迫的响应强烈。本研究的结果表明,NaNHX1基因属于Na+/H+逆向转运蛋白家族,可能参与了花烟草高盐和低钾胁迫,以及其它非生物胁迫响应在内的众多生理过程。     

花烟草NaERF1基因的克隆及在非生物胁迫下的表达模式分析

AP2/ERF类转录因子,是植物所特有的最大的一类转录因子家族,在植物的生长发育过程中,扮演着重要的角色。探究花烟草ERF转录因子的生理功能,为花烟草抵御逆境的分子机制研究提供借鉴。采用同源克隆的方法进行基因克隆。通过对花烟草进行非生物胁迫,运用qPCR的方法进行基因表达模式分析。从花烟草(Nicotiana alata)中克隆了一个属于ERF家族的基因NaERF1。该基因的开放阅读框全长为819 bp,编码了272个氨基酸。生物信息学分析结果表明,该基因编码的蛋白分子量为30.7 kD,等电点为6.07;具有AP2/ERF类转录因子家族典型的保守结构域;该基因主要定位于细胞质内,并含有多个磷酸化位点。同源性分析的结果显示,NaERF1基因与茄科植物的ERF同源性较高,并且与普通烟草的ERF亲缘关系最近。NaERF1基因的表达具有组织表达特异性,花中表达量最高,茎中次之,根和叶中表达量较低。同时,在高盐、干旱、低温、ABA、低钾及H2O2等非生物胁迫下,NaERF1的表达呈现5种模式。其中,对低钾及ABA胁迫的响应强烈。NaERF1基因属于AP2/ERF类转录因子,可能广泛参与了花烟草包括非生物胁迫响应在内的众多生理过程。     

费尔干猪毛菜病程相关蛋白基因SfPR-1的表达规律和植物表达载体构建

采用RT-PCR技术探究盐生植物费尔干猪毛菜病程相关蛋白基因SfPR-1(GenBank登录号:JQ670917)在不同发育时期、组织部位、植物激素、非生物胁迫及生物胁迫处理下的表达规律,以揭示该基因在费尔干猪毛菜生长发育和逆境胁迫下的作用。结果表明,不同组织(根、茎、叶)中,SfPR-1在根中表达量最高,预示该基因可能在根防御中发挥主要作用;SfPR-1在不同发育阶段(种子、种子萌发20 d幼苗、种子萌发30 d幼苗、种子萌发40 d幼苗)的表达特性显示,种子萌发30 d幼苗时,其表达差异最显著,表明其可能在植株后期生长发育中具有重要作用;SfPR-1对不同植物激素(水杨酸SA、茉莉酸JA、脱落酸ABA、乙烯合成前体ACC)均产生了响应,表明该基因在几条主要的抗病防御通路中均发挥作用。非生物胁迫(H2O2、盐、旱、冷)都能够诱导SfPR-1基因的表达,其中对盐响应程度最高。以植源性意大利青霉(Penicillium italicum)进行生物胁迫时,SfPR-1表达呈持续上升趋势。以上结果表明费尔干猪毛菜病程相关蛋白基因SfPR-1是生物与非生物逆境胁迫下均响应的基因,推测其在植物抵御逆境胁迫中发挥重要作用。     

紫花苜蓿NAC转录因子MsNAC1基因的克隆、生物信息学分析及非生物逆境胁迫下的表达分析

NAC转录因子是高等植物所特有的具有多种生物功能的转录因子,在植物生长发育、抵抗逆境和激素调节等过程中发挥着重要作用。本研究利用RACE-PCR技术,克隆获得了紫花苜蓿NAC转录因子Ms NAC1(Gen Bank登录号为JN099384.1)基因的c DNA序列。生物信息学分析显示,Ms NAC1基因的开放阅读框(ORF)为993 bp,编码一个由330个氨基酸残基组成的亲水性蛋白,N-端具有保守的NAM结构域,C-端高度变异,具备NAC转录因子的基本特征;Ms NAC1蛋白被定位在细胞核中,含有2条核定位信号序列,具有9个糖基化位点和23个磷酸化位点,三级结构为对称的同型二聚体。多重比对发现,Ms NAC1蛋白与拟南芥ATAF1和水稻Os NAC6蛋白的同源性较高;系统进化分析表明,Ms NAC1蛋白属于NAC转录因子家族中的ATAF亚族,与ATAF1的亲缘关系最近。非生物逆境胁迫下的表达分析显示,Ms NAC1基因在高盐、干旱和低温胁迫下表达量均呈现先上调后下调的趋势,不同处理时间的差异达到极显著水平,并且根中的表达量上调幅度高于叶片,说明该基因可能参与调控非生物逆境胁迫的生理响应。     

青杆PwUSP1基因的克隆及表达模式分析

广泛逆境胁迫蛋白（universalstressprotein,USP）在非生物胁迫响应中起重要作用,但在植物中其功能还大部分未知。本研究通过BLAST分析青杆EST文库,得到职zP基因的EST序列,通过RACEPCR方法获取USP基因的末端序列,经过与EST序列拼接得到USP基因的cDNA全长序列,命名为PwUSP1。分析发现PwUSP1全长cDNA为1167bp,编码区为519bp,编码172个氨基酸残基。生物信息学分析显示,PwUSP1编码的蛋白相对分子质量为19．07kDa,理论等电点为6．38,为非跨膜的亲水性蛋白。PwUSP1具有USP家族典型的UspA结构域和ATP结合位点G-（2x）-G-（9x）-G（S／T）。半定量RT-PCR与RT-qPCR分析表明,PwUSP1在青杆的根、茎、针叶、花粉、种子中均有表达,在根和花粉中表达量较高。同时,PwUSP1受干旱和盐胁迫的诱导表达上调,均在处理6h后表达量较高,推测该基因可能在青杆逆境胁迫响应中发挥作用。     

柠条锦鸡儿CkLEA1基因克隆及表达分析

植物受到逆境胁迫后,大量逆境响应基因会被诱导表达,LEA蛋白编码基因就是与植物抗旱、抗冷等非生物胁迫密切相关的一类基因.从已构建的柠条锦鸡儿干旱胁迫抑制性削减杂交文库中筛选到了一条LEA蛋白编码基因并进行了克隆.序列比对与系统进化分析显示该基因属于LEA3基因家族成员,命名为CkLEA1(GenBank登录号是KC309408).克隆得到该基因gDNA长469bp,包含两个外显子和一个内含子;cDNA长357bp,包含300bp的开放阅读框,推导编码99个氨基酸的蛋白质.利用荧光定量PCR技术对CkLEA1基因在各种逆境胁迫条件的表达情况进行初步研究表明,CkLEA1受干旱、ABA、冷、热、盐和碱等处理不同程度地诱导,推测其与柠条锦鸡儿响应逆境胁迫的机制有关.     

高羊茅MAPK基因的克隆与表达分析

丝裂原活化蛋白激酶(M APK)是生物体内信号转导的重要组分,与生长、发育和逆境胁迫反应密切相关.为了研究草坪草对非生物逆境胁迫反应的分子机理,利用同源基因克隆法从4℃低温诱导的草坪草高羊茅(F estu-ca arund inacea Schreb.)幼苗cDNA文库中分离得到一个M APK的cDNA即F aMAPK 1,F aMAPK 1编码369个氨基酸残基的蛋白激酶,该蛋白激酶具有TEY的磷酸化基序.据推测的氨基酸序列的BLA ST同源性分析表明,F aM APK 1蛋白与水稻O sM APK 4蛋白的一致性为91.1%.N orthern杂交检测F aMAPK 1基因对逆境胁迫反应的结果表明冷(4℃)处理对根中F aMAPK 1基因的表达没有明显影响,但诱导叶中F aMAPK 1上调表达.而且低温(4℃)、高盐(250 mm o l/L N aC l)、干旱和100μm o l/L ABA都诱导叶中F aMAPK 1上调表达,表明F aM APK 1蛋白可能在高羊茅对非生物逆境胁迫的反应中起重要作用.     

陆地棉干旱胁迫相关基因GhPR-10的克隆及表达分析

病程相关蛋白(pathogenesis-related proteins,PR)的产生与积累是植物体应对生物或非生物胁迫的主要特征之一,而病程相关蛋白10(PR-10)是这类蛋白家族中的重要成员。该研究在前期蛋白质组学分析的基础上,采用同源克隆法和RACE技术相结合,设计了一对引物,克隆了陆地棉"KK1543"干旱胁迫下差异表达的Gh PR-10。Gh PR-10基因具有一个432 bp的开放阅读框,编码144个氨基酸,预测分子量约为15.6 k D,等电点为4.90。氨基酸序列比对和同源性分析显示Gh PR-10蛋白与其他植物中的PR-10蛋白有较高的一致性,说明该基因在进化过程中是相当保守的。系统进化树分析显示,陆地棉Gh PR-10与海岛棉、雷蒙德氏棉中蛋白的亲缘关系最近。为了研究不同胁迫条件和不同组织器官该基因的表达情况,采用荧光定量PCR技术,研究表明在15%PEG胁迫下,Gh PR-10在根中同时期的表达量大部分都比在茎和叶同时期的表达量要高;Gh PR-10的表达量在3种胁迫下变化趋势整体相似,且都呈现上调表达,干旱和盐胁迫下该基因的表达量在24 h达到最高,4℃低温胁迫下该基因在12 h达到最高。上述实验表明,Gh PR-10基因可能参与棉花防御逆境胁迫机制,为进一步研究Gh PR-10基因功能奠定了一定的基础。     

CIPK7 is involved in cold response by interacting with CBL1 in Arabidopsis thaliana

The family of calcineurin B-like (CBL) proteins is a unique group of Ca²⁺ sensors in plants. CBLs relay the calcium signal by interacting with and regulating the family of CBL-interacting protein kinases (CIPKs). Extensive studies have demonstrated that the CBL-CIPK complexes mediate plant responses to a variety of external stresses. However, there are few reports on the CBL-CIPK involved in cold stress responses. In this study, we analyzed expression of CIPK7 and CBL1 in Arabidopsis during cold treatments. Expression of CIPK7 was induced by cold, and CIPK7 interacted with CBL1 in vitro. Moreover, affinity chromatography purification of CIPK7 from Arabidopsis plants using CBL1 suggested that CIPK7 may associate with CBL1 in vivo. Expression of CBL1 was cold inducible, and CBL1 had a role in regulating cold response. By comparing expression patterns of CIPK7 between wild-type and cbl1 mutant plants, we found the induction of CIPK7 by cold stress was influenced by CBL1. This is the first report to demonstrate that CIPK7 may play a role in cold response via its interaction with CBL1.     

Nylon Filter Arrays Reveal Differential Expression of Expressed Sequence Tags in Wheat Roots Under Aluminum Stress

To enrich differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) nearisogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to Al stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg2 transportation, and other functions. Under Al stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to Al stress may involve complicated defense-related signaling and metabolic pathways.The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress.     

CBL1, a calcium sensor that differentially regulates salt,drought, and cold responses in Arabidopsis

Although calcium is a critical component in the signal transduction pathways that lead to stress gene expression in higher plants, little is known about the molecular mechanism underlying calcium function. It is believed that cellular calcium changes are perceived by sensor molecules, including calcium binding proteins. The calcineurin B-like (CBL) protein family represents a unique group of calcium sensors in plants. A member of the family, CBL1, is highly inducible by multiple stress signals, implicating CBL1 in stress response pathways. When the CBL1 protein level was increased in transgenic Arabidopsis plants, it altered the stress response pathways in these plants. Although drought-induced gene expression was enhanced, gene induction by cold was inhibited. In addition, CBL1-overexpressing plants showed enhanced tolerance to salt and drought but reduced tolerance to freezing. By contrast, cbl1 null mutant plants showed enhanced cold induction and reduced drought induction of stress genes. The mutant plants displayed less tolerance to salt and drought but enhanced tolerance to freezing. These studies suggest that CBL1 functions as a positive regulator of salt and drought responses and a negative regulator of cold response in plants.     

Nylon Filter Arrays Reveal Differential Expression of Expressed Sequence Tags in Wheat Roots Under Aluminum Stress

To enrich differentially expressed sequence tags (ESTs) for aluminum (A1) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) nearisogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using suppression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to A1 stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg^2 transportation, and other functions. Under A1 stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to A1 stress may involve complicated defense-related signaling and metabolic pathways. The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress.     

Expression of the Tobacco Ext 1.4 Extensin Gene Upon Mechanical Constraint and Localization of Regulatory Regions

Abstract: The regulation of the Ext 1.4 gene encoding a tobacco ( Nicotiana tabacum L.) extensin was studied in response to mechanical constraints. Transgenic plants carrying chimeric Ext 1.4 promoter/GUS (β-glucuronidase)/ nos terminator or Ext 1.4 3'-end constructs were obtained. Expression of gene fusions was found in tissues where mechanical stresses occur, e.g., during germination, as well as in root and stem tissues. Chimeric genes were successively and transiently expressed in different tissues during germination, i.e., at the tip of the root and then in the hypocotyl, during their growth through the seed coat. Moreover, they were expressed in cortical cells surrounding the emergence of adventitious and lateral roots and developmentally-regulated in nodes. The expression of Ext 1.4 could be induced by imposing mechanical constraints due to curving of either the stems or roots. Expression then occurred in cells where it does not normally occur, i.e., in cortical cells of internodes and in the distal piliferous zone of roots. Accumulation of RNAs occurs several days after the start of the constraint. Promoter regions involved in regulation of expression of Ext 1.4 in stems, roots, and in seedlings upon mechanical constraint could be localized. Moreover, the 3' non-coding region was shown to modulate expression in roots. These results suggest that the regulation of Ext 1.4 following mechanical stress is dependent on both tissue-specific and mechanical-responsive elements.     

Localization and control of expression of Nt-Syr1, a tobacco SNARE protein

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion and secretion. Previously, we isolated the syntaxin-related protein Nt-Syr1 from Nicotiana in a screen for ABA-related signalling elements, and demonstrated its role in determining the ABA sensitivity of stomatal guard cells. Because the location and expression of SNAREs are often important clues to their functioning, we have examined the distribution and stimulus-dependent expression of Nt-Syr1 between tissues, as well as its location within the cell, using antisera raised against purified recombinant peptides corresponding to overlapping cytosolic domains of Nt-Syr1. The Nt-Syr1 epitope was strongly represented in roots and to lesser extents in stems, leaves and flowers of well-watered plants. Biochemical analysis and examination of immunogold labelling under the electron microscope indicated Nt-Syr1 to be located primarily at the plasma membrane. Expression of the protein in leaves and to a lesser extent in flowers and stems was transiently enhanced by ABA, but not by auxin, kinetin or gibberellic acid. Expression in leaves was promoted by salt stress and wounding, but not by cold. By contrast, Nt-Syr1 levels in the root were unaffected by ABA. In the leaves, enhanced expression of Nt-Syr1 by salt stress was not observed in aba1 mutant Nicotiana, which is deficient in ABA synthesis, and in plants carrying the Arabidopsis abi1 transgene that suppresses a number of ABA-evoked responses in these plants. However, an enhanced expression in response to wounding was observed, even in the mutant backgrounds. We conclude that Nt-Syr1 expression at the plasma membrane is important for its function and is subject to control by parallel, stress-related signalling pathways, both dependent on and independent of ABA. Nt-Syr1 may be associated with additional functions, especially in the roots, that are unrelated to ABA or stress responses in the plant.