壳寡糖诱导植物抗性相关基因mRNA差别显示分析

壳寡糖是一种天然的信号分子,可以诱导植物产生各种防御反应。该文利用mRNA显示技术。研究壳寡糖引起的植物抗性相关基因的变化。以50mg/l壳寡糖喷洒的烟草叶片为材料,分别提取处理8h、7d和对照的叶片总RNA,利用一个锚定引物和三个随机引物组合,通过mRNA差别显示技术共获得壳寡糖处理后发生变化的8h差别条带22条,7d后的差别条带为74条。将壳寡糖处理8h得到的17条差别条带克隆到载体上后,进行反向Northern鉴定,有6条可能为真实条带。测序结果表明：其中一条差别片段与烟草热激蛋白90基因具有97％的核苷酸同源性。说明50mg/l壳寡糖处理的烟草叶片在mRNA水平上发生了明显的变化,烟草的热激蛋白90基因可能参与到壳寡糖诱导的抗性信号传导通路中。     

NtSKP1基因的反义载体构建及转基因烟草的产生

根据枯斑三生烟SKP1基因(NtSKP1)的序列,设计一对分别含有特定酶切位点的特异引物,以重组质粒pMD18-SKP1为模板,扩增目的基因(约473bp)片段。将反向目的片段插入中间载体pHANNIBAL的内含子右侧,再经NotⅠ酶切回收约3443bp的目的片段,插入到双元载体质粒pART27中,成功构建了含NtSKP1基因片段反向序列的植物表达载体pART27-skp1a,其转录产物能减弱目的基因的表达。将pART27-skp1a质粒导人根癌农杆菌LBA4404中并转化烟草叶片细胞,经选择分化培养,获得转基因烟草。     

壳寡糖对烟草悬浮细胞茉莉酸合成基因转录的影响

采用RT-PCR方法研究了不同浓度壳寡糖对烟草悬浮细胞茉莉酸合成酶基因的转录调控。结果表明, 50 μg.mL-1壳寡糖能够明显诱导烟草悬浮细胞茉莉酸合成途径的关键酶——磷脂酶A2、13-脂氧合酶、丙二烯氧化物合成酶、丙二烯氧化物环化酶和12-氧-植物二烯酸还原酶基因的表达, 而且该浓度的壳寡糖对这些基因的诱导作用相同(似)。在实验设定时间内均诱导表达编码磷脂酶A2的基因, 对其它基因的诱导时间均为8小时, 表明50 μg.mL-1壳寡糖在诱抗过程中启动了茉莉酸合成途径。而200 μg.mL-1壳寡糖的处理对这些基因的表达无显著影响。表明不同浓度的壳寡糖对烟草悬浮细胞的作用模式存在差异, 且高浓度的壳寡糖在烟草悬浮细胞中启动的信号通路可能没有茉莉酸信号的参与。     

烟草SKP1基因的原核表达

为获取大量高纯度的枯斑三生烟SKP1蛋白以便研究其功能和性质,以pMD18-SKP1质粒为模板, PCR扩增枯斑三生烟的SKP1基因的cDNA编码区,经酶切后构建表达载体pET23b-SKP1,转入大肠杆菌BL21(DE3)进行诱导表达,并考察不同的IPTG诱导终浓度、表达时间和表达温度对目的蛋白His-SKP1表达量的影响.SDS-PAGE分析结果表明:最佳表达条件为不加诱导剂的情况下,37℃表达8 h.     

壳寡糖对烟草悬浮细胞茉莉酸合成基因转录的影响

采用RT-PCR方法研究了不同浓度壳寡糖对烟草悬浮细胞茉莉酸合成酶基因的转录调控。结果表明,50μg·mL^-1壳寡糖能够明显诱导烟草悬浮细胞茉莉酸合成途径的关键酶——磷脂酶A2、13-脂氧合酶、丙二烯氧化物合成酶、丙二烯氧化物环化酶和12-氧-植物二烯酸还原酶基因的表达,而且该浓度的壳寡糖对这些基因的诱导作用相同（似）。在实验设定时间内均诱导表达编码磷脂酶A2的基因,对其它基因的诱导时间均为8小时,表明50μg·mL^-1壳寡糖在诱抗过程中启动了茉莉酸合成途径。而200μg·mL^-1壳寡糖的处理对这些基因的表达无显著影响。表明不同浓度的壳寡糖对烟草悬浮细胞的作用模式存在差异,且高浓度的壳寡糖在烟草悬浮细胞中启动的信号通路可能没有茉莉酸信号的参与。     

激发子基因peaT1转化三生烟及其对TMV抗性的提高

通过根癌农杆菌(Agrobactrium tumefaciens)介导转化法,将含有激发子基因peaT1的植物表达载体pCAM-BIA2300-G4AS-peaT1转化三生烟,获得了转基因烟草植株。用PCR检测确认了阳性转化株,用Southern杂交、RT-PCR和Western杂交进一步证实了peaT1基因的整合、转录和表达。对T1代转基因阳性株进行TMV接种试验,结果显示,与非转基因对照相比,表达peaT1的烟草叶片枯斑数量减少,表明蛋白激发子基因peaT1的表达提高了转基因烟草对TMV的抗性。     

壳寡糖诱导植物防御反应中一氧化氮信号的研究

壳寡糖可以增强植物对病虫害的防御能力,为了深入研究壳寡糖的作用机理,首次运用荧光酶标仪及一氧化氮(Nitric oxide,NO)荧光探针Diaminofluorescein diacetate (DAF-2DA)对壳寡糖诱导的NO信号进行研究。研究发现,不同浓度的壳寡糖均可诱导烟草悬浮细胞产生NO;NO的清除剂Carboxy-PTIO potassium salt(cPTIO)和一氧化氮合酶(Nitric oxide synthase,NOS)抑制剂N^ω-nitro-L-arginine methyl Ester(L-NAME)可以明显抑制NO的产生;硝酸还原酶(Nitrate reductase, NR)的抑制剂叠氮化钠和钨酸钠对NO的产生无影响;Ca²⁺流相关抑制剂氯化镧和钌红均可抑制NO的产生。NO和Ca²⁺流的相关抑制剂可明显抑制壳寡糖诱导的抗性相关基因的表达。结果显示:壳寡糖主要通过NOS酶催化合成NO,且NO参与调节壳寡糖诱导的抗性相关基因的表达,在此过程中,Ca²⁺可以调节NO的合成。     

SPINDLY在壳寡糖诱导拟南芥抗丁香假单胞菌中的功能

为了探讨拟南芥O-岩藻糖基转移酶(SPINDLY)在病原体相关分子模式诱导抗性中的作用,该研究以SPINDLY缺失拟南芥突变体spy-3为实验材料,从叶片表型、病情指数、病菌定殖量以及丁香假单胞菌(Pst DC3000)关键基因的表达水平等指标,系统考察了SPINDLY在壳寡糖诱导拟南芥抗Pst DC3000中的功能。结果显示:(1)spy-3突变体比野生型更易被Pst DC3000侵染。(2)与病菌侵染组相比,壳寡糖预处理明显缓解植株叶片黄化现象,显著降低Pst DC3000的定殖量。(3)壳寡糖预处理的spy-3植株中水杨酸和茉莉酸途径相关基因的表达量及水杨酸和茉莉酸含量均较病菌侵染组明显升高。(4)壳寡糖在spy-3中的诱抗效果与野生型相比无明显差别。研究表明,SPINDLY在植物先天免疫过程发挥重要作用,但并不影响壳寡糖的诱导抗性。     

壳寡糖诱导烟草对TMV长距离移动的影响

采用ELISA-DSM法和半叶枯斑法,测定了壳寡糖(50μg/mL)诱导后普通烟(Nicotiana tabacum)植株体内TMV浓度的变化.ELISA-DSM测定显示,在接种后10 d,仅在接种叶的上位叶和新生叶片中检测到病毒,且病毒浓度仅为不诱导对照的52.7%和38.8%,在下位叶中未检测到病毒;同时,接种叶内病毒增殖严重受抑,接种后10 d,病毒浓度仅为不诱导对照的23.52%.半叶枯斑法检测获得了相同结果,以壳寡糖处理植株的不同叶位的叶片为毒源,产生的枯斑数目都大幅度低于不诱导对照.以上结果证明,壳寡糖处理后TMV的上行和下行长距离移动均明显延迟和减少,下行移动受到的影响更大.透射电镜检查发现,处理植株接种叶的下位叶片韧皮部细胞中没有病毒晶体和病毒粒子,在上位叶片筛管伴胞中仅见少量病毒粒子,两者都未发现任何诱导新生物,也未见其他细胞结构变化.结果表明,壳寡糖处理使烟草对TMV病毒侵染产生了诱导抗病性,系统侵染症状明显减弱;壳寡糖处理对病毒长距离移动的不利影响可能是接种叶片病毒增殖减少所造成的.     

真菌寡糖诱导植物抗性活性成分的分离纯化

寡聚糖作为一种信号分子 ,在调节植物的生长、发育以及植物在不同环境中生存能力等方面起着非常重要的作用[1] .许多特定结构的寡糖被证明具有诱导植物抗性的作用 .对具有诱导抗性作用的葡聚寡糖结构分析表明 ,其最小活性寡糖单位是由 7个葡葡糖残基组成的 β 葡聚糖苷 ,在一个     

Several components of SKP1/Cullin/F-box E3 ubiquitin ligase complex and associated factors play a role in Agrobacterium-mediated plant transformation

? Successful genetic transformation of plants by Agrobacterium tumefaciens requires the import of bacterial T-DNA and virulence proteins into the plant cell that eventually form a complex (T-complex). The essential components of the T-complex include the single stranded T-DNA, bacterial virulence proteins (VirD2, VirE2, VirE3 and VirF) and associated host proteins that facilitate the transfer and integration of T-DNA. The removal of the proteins from the T-complex is likely achieved by targeted proteolysis mediated by VirF and the plant ubiquitin proteasome complex. ? We evaluated the involvement of the host SKP1/culin/F-box (SCF)-E3 ligase complex and its role in plant transformation. Gene silencing, mutant screening and gene expression studies suggested that the Arabidopsis homologs of yeast SKP1 (suppressor of kinetochore protein 1) protein, ASK1 and ASK2, are required for Agrobacterium-mediated plant transformation. ? We identified the role for SGT1b (suppressor of the G2 allele of SKP1), an accessory protein that associates with SCF-complex, in plant transformation. We also report the differential expression of many genes that encode F-box motif containing SKP1-interacting proteins (SKIP) upon Agrobacterium infection. ? We speculate that these SKIP genes could encode the plant specific F-box proteins that target the T-complex associated proteins for polyubiquitination and subsequent degradation by the 26S proteasome.     

Patterns of gene duplication in the plant SKP1 gene family in angiosperms: evidence for multiple mechanisms of rapid gene birth

Gene duplication plays important roles in organismal evolution, because duplicate genes provide raw materials for the evolution of mechanisms controlling physiological and/or morphological novelties. Gene duplication can occur via several mechanisms, including segmental duplication, tandem duplication and retroposition. Although segmental and tandem duplications have been found to be important for the expansion of a number of multigene families, the contribution of retroposition is not clear. Here we show that plant SKP1 genes have evolved by multiple duplication events from a single ancestral copy in the most recent common ancestor (MRCA) of eudicots and monocots, resulting in 19 ASK (Arabidopsis SKP1-like) and 28 OSK (Oryza SKP1-like) genes. The estimated birth rates are more than ten times the average rate of gene duplication, and are even higher than that of other rapidly duplicating plant genes, such as type I MADS box genes, R genes, and genes encoding receptor-like kinases. Further analyses suggest that a relatively large proportion of the duplication events may be explained by tandem duplication, but few, if any, are likely to be due to segmental duplication. In addition, by mapping the gain/loss of a specific intron on gene phylogenies, and by searching for the features that characterize retrogenes/retrosequences, we show that retroposition is an important mechanism for expansion of the plant SKP1 gene family. Specifically, we propose that two and three ancient retroposition events occurred in lineages leading to Arabidopsis and rice, respectively, followed by repeated tandem duplications and chromosome rearrangements. Our study represents a thorough investigation showing that retroposition can play an important role in the evolution of a plant gene family whose members do not encode mobile elements.     

小麦几丁质酶基因的异种表达及其功能鉴定

几丁质酶参与植物的发育及防卫反应,并与人类疾病发生有关.文章研究了小麦几丁质酶基因Wch2经根癌农杆菌介导的烟草瞬间表达和转基因拟南芥的稳定表达,Western杂交及酶活测定证实,瞬间表达的小麦几丁质酶分子量约30 kD,具有降解几丁质多聚物的功能;Wch2在转入拟南芥后表达量高,尖孢镰刀菌接种的鉴定表明,表达Wch2的转基因植株的抗病性显著高于表达绿色荧光蛋白的对照植株.这些结果说明Wch2的异种表达,可用于植物抗病基因工程,以增强植物的抗病性.     

不同启动子控制下Ac转座酶基因的表达对玉米Ds因子在水稻中切离频率的影响

用水稻愈伤组织比较了Ac启动子、35S启动子与Ubi启动子控制下Ac转座酶基因（Ts）的表达对Ds因子切离频率的影响。结果表明Ubi启动子与Ac转座酶编码区嵌合基因（Ubipro-Ts）反式激活Ds因子的切离频率最高,达到了72.9%。通过杂交将Ubipro-Ts基因导入Ds因子转化植株,得到9株Ubipro-Ts基因与Ds因子共存的F1代杂交水稻植株,其中有8株Ds因子发生了切离。用Inverse-PCR的方法从其中一株杂交植株中克隆到Ds因子的旁邻序列,其DNA顺序与亲本中Ds因子原插入位点的序列不同,表明Ds因子转座到了新的基因组位点。     

小麦3个被白粉菌诱导基因表达的分析

含有抗白粉病基因Pm2 1的小麦 簇毛麦 6VS/6AL易位系在接种白粉菌后 ,叶片无任何病症。应用mRNA差异显示技术从小麦 簇毛麦 6VS/ 6AL易位系分离到 3个叶绿体蛋白基因片段 ,它们是TaD5、TaD2 3和TaD33,3个基因片段分别与小麦叶绿体基因rbcL ,拟斯卑尔脱山羊草叶绿体RNA聚合酶α亚基基因rpoA和大麦 1,5 二磷酸核酮糖羧化酶活化酶基因(Rubiscoactivase ,RcaA2 )同源性达 97%、98%和 88%。据此推测TaD5、TaD2 3和TaD33分别是 6VS/ 6AL易位系中的rbcL、rpoA和 1,5 二磷酸核酮糖羧化酶活化酶基因的片断。Northern分析表明这 3个叶绿体基因的表达在白粉菌诱导下得到增强。叶绿体基因组含有胸腺嘧啶重复区是在mRNA差异显示中克隆到叶绿体基因组基因的原因     

Cloning and characterization of a non-TIR-NBS-LRR type disease resistance gene analogue from peach.

Cloning of plant disease resistant genes is greatly helpful for disease resistant breeding in plants and the insight of resistance mechanism. However, there are less relevant researches in peach [prunus persica (L.) Batch]. In this study, four NBS-LRR type resistance gene analogs (RGAs) were cloned from genomic DNA of peach. The PNBS2 fragment was also amplified from peach cDNA and the full-length cDNA of PNBS2 (PRPM1, GenBank accession no. AY599223) has been cloned. Sequence analysis indicated that the cDNA of PRPM1 is 3007 bp in length and that the contained ORF encodes for a polypeptide of 917 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has typical structure of non-TIR-NBS-LRR genes, with NB-ARC, LZ, LRR and transmembrane domains. Southern analysis indicated that the PRPM1 gene might be a single copy in peach genome. Northern blot and RT-PCR analysis showed that the expression of PRPM1 was not induced by salicylic acid (SA) in peach young leaves. The isolation of putative resistance genes from peach provided useful bases for studying the structure and function of peach disease-resistance relating genes and disease resistant genetic breeding in peach.     

ERF类转录因子OPBP1基因的超表达提高烟草的耐盐能力

ERF是植物中的一类重要的转录因子,参与调节植物的生长,发育以及抗胁迫等过程,对一烟草OPBP1基因（属于ERF类基因）的烟草转化,获得了该基因超表达的植株,转基因植株明显地增加了耐盐能力,Northern杂交结果表明,OPBP1基因有不同程度的表达,而且表达丰度与其耐盐性有一定的正相关性,凝胶阻滞实验结果证明OPBP1融合蛋白能特异地与含GCC盒的DNA序列结合,这些结果说明OPBP1基因可能作为一转录因子来调节烟草耐盐相关的基因。     

水稻MYB cDNA的克隆和表达分析

根据植物MYB类转录因子DNA结合功能域的保守区设计一对简并引物 ,以水稻根、小苗和未成熟种子中的RNA为材料 ,用RT PCR方法扩增出约 180bp的片段。序列分析表明 ,它们与MYB基因的保守区有很好的同源性。以未成熟种子中获得的这一 180bp片段作探针 ,从水稻未成熟种子cDNA文库中分离到 5个新的MYB基因家族成员 ,它们是OsMYB12、13、14、15和5 1。在酵母系统中证实OsMYB13、OsMYB15和Os MYB5 1蛋白具有转录激活功能。Northern印迹分析表明 ,OsMYB5 1主要在未成熟种子中表达 ,在根和小苗中表达水平较低。RT PCR分析表明 ,OsMYB15在根、茎、小穗、叶片和种子中有低水平的表达     

Structural,Expression and Interaction Analysis of Rice SKP1-Like Genes

The degradation of proteins by the 26S proteasome is initiated by protein polyubiquitination mediated by a three-step cascade. The specific ubiquitination of different target proteins is mediated by different classes of E3 ubiquitin ligases, among which the best known are Skp1-Cullin-F-box complexes. Whereas protists, fungi and some vertebrates have a single SKP1 gene, many animal and plant species possess multiple SKP1 homologues. In this paper, we report on the structure, phylogeny and expression of the complete set of rice SKP1 genes (OSKs, Oryza sativa SKP1-like genes). Our analyses indicated that OSK1 and OSK20 belong to a class of SKP1 genes that contain one intron at a conserved position and are highly expressed. In addition, our yeast two-hybrid results revealed that OSK proteins display a differing ability to interact with F-box proteins. However, OSK1 and OSK20 seemed to interact with most of the nine F-box proteins tested. We suggest that rice OSK1 and OSK20 are likely to have functions similar to the Arabidopsis ASK1 and ASK2 genes.