西瓜APX基因的序列分析及其茉莉酸甲酯诱导表达特性

抗坏血酸过氧化物酶(ascorbate peroxidase,APX)作为植物抗氧化防御系统中的重要一员,在一定程度上决定着植物的抗逆性。茉莉酸甲酯(methyl jasmonate,Me JA)作为逆境信号物质可以诱导植物抗逆响应,但是能否诱导APX基因表达、其表达趋势如何到目前为止尚未见报道。我们根据拟南芥APX同源基因At APXIII(AJ006030.1)的序列在西瓜基因组数据库中搜索获得一个高度同源序列:Cla015833,命名为Cl APX1。我们对该基因进行了生物信息学分析并以二倍体西瓜细胞为材料,利用q RT-PCR技术对Cl APX1在茉莉酸甲酯诱导下的表达特性进行了研究。结果表明Cl APX1基因的转录起始位点位于起始密码子上游980 bp处,启动子区域包含典型启动子必须的调控元件,多个激素响应元件和逆境胁迫响应元件等;该基因编码的蛋白含有286个氨基酸,分子量为31.56 k D,理论等电点为6.67,此蛋白可能定位在细胞质中,属于c APX;该基因在亲缘关系上较为接近同科的黄瓜和甜瓜。在茉莉酸甲酯施加了0.5 h后Cl APX1基因的表达量高于本底水平,一直持续到8 h,变化呈现出先上升后下降的趋势。本研究说明了Cl APX1基因对茉莉酸甲酯模拟的逆境信号做出了响应且表达量提高。本研究为利用茉莉酸甲酯提高植物抗逆性提供了理论支持,为植物的APX基因对茉莉酸信号响应方式和表达调控趋势做了补充,为以后深入研究APX基因奠定基础。     

丹参miR408基因前体序列的克隆及表达分析

MiR408是植物中一类高度保守的miRNA,其靶基因编码含铜蛋白,miR408的表达受植物生长发育和环境条件的显著影响。拟南芥中miR408在HY5-SPL7基因网络中起着关键作用,而HY5-SPL7基因网络可介导拟南芥对光照和铜离子的协调应答,进一步表明miR408在植物对环境的响应中发挥了核心作用。本研究基于丹参基因组Survey数据库,从中搜索并克隆得到366 bp的含有稳定茎环结构的miR408前体序列及其上游723 bp片段的启动子区,Gen Bank登录号分别为KU360384和KU360385,并将miR408的前体序列命名为SmMIR408。生物信息学分析结果显示:Sm-MIR408上游启动子序列与模式植物拟南芥Ath-MIR408、水稻OsaMIR408启动子区具有许多相同的顺式作用元件,如G-box、CGTCA-motif、TGACG-motif、GTAC-motif等,而HSE、CATT-motif作用元件仅存在于丹参启动子区;miR408成熟序列在不同物种中具有很高的保守性;基于不同物种miR408前体序列构建的系统进化树显示,来源于单子叶植物的miR408前体序列聚为一支,来源于双子叶植物的miR408前体序列聚为另一支。实时定量PCR分析结果显示:Sm-MIR408在丹参的根、茎、叶、花中都有表达,其在花中的表达量最高,根中最低;Sm-MIR408的表达受茉莉酸甲酯和伤害的抑制,黑暗和持续光照也可抑制该基因的表达。Sm-MIR408基因的克隆与表达分析为今后研究丹参miR408的功能奠定了基础。     

苹果果实β-半乳糖苷酶基因启动子的克隆与功能分析

根据已发表的苹果基因组序列设计特异引物,克隆得到苹果品种‘嘎拉’中β-半乳糖苷酶基因(Md-gal)的启动子。序列分析表明,该启动子除含有大多数高等植物启动子具有的保守元件外,还含有大量光响应元件和与激素相关的顺式作用元件,主要有赤霉素响应元件(GARE-motif和P-box)、茉莉酸甲酯(MeJA)应答元件(CGTCA-motif和TGACG-motif)和生长素响应元件(TGA-element);构建5个不同长度Md-gal启动子和GUS基因融合的瞬时表达载体,并进行苹果果实和烟草叶片转化试验,结果显示,5个启动子均具有启动子活性,在Md-gal启动子序列中激活与抑制调节元件并存,正调控元件位于-1206~-754bp区域,负调控元件位于-754~-597bp区域;对Md-gal启动子进行激素响应分析结果显示,激素处理可对其产生诱导作用,且MeJA处理后GUS活性最高。研究表明,Md-gal启动子具有真核基因启动子的基本结构特征和正负调控特性,可响应外源激素处理,可能在苹果果实生长发育和成熟软化方面具有一定的作用。     

青杄生长素抑制蛋白基因PwARP-1的克隆及表达分析

从青杄的cDNA文库中筛选出一个生长素抑制蛋白基因PwARP-1(Auxin-repressed protein 1),并通过RACE-PCR技术获得PwARP-1 cDNA全长。生物信息学分析显示,PwARP-1基因编码155个氨基酸残基的蛋白,分子量为17.1 kD,理论等电点为10.07,富含无规则卷曲。以多年生青杄和青杄幼苗为研究材料,通过RT-qPCR技术检测PwARP-1的组织特异性表达、激素的响应模式及种子萌发过程中的表达量模式。结果表明,PwARP-1在青杄各个组织中均有表达,在花粉中表达量最高,在种子中的表达量最低。PwARP-1在种子萌发过程中表达量呈先上升,在第10天表达量达到最高后开始下降。进一步试验表明,PwARP-1能响应多种逆境胁迫和激素处理。在赤霉素(GA)和生长素(IAA)处理下PwARP-1的表达量被抑制。在干旱胁迫和油菜素内酯(BR)激素处理下PwARP-1的表达量逐渐升高,而在在盐胁迫、茉莉酸甲酯(MeJA)和脱落酸(ABA)等激素处理下PwARP-1的表达量先下降后上升。这些结果显示PwARP-1可能参与了植物种子萌发、多种逆境胁迫和激素响应过程。     

OsEBP-89启动子中应答茉莉素信号的必需DNA区域的分析

水稻OsEBP-89基因的表达受乙烯（ET）、脱落酸（ABA）、茉莉素等激素和干旱、低温等逆境胁迫处理的诱导．本研究中,克隆该基因启动子和预测应答胁迫与激素信号相关顺式作用元件的基础上,通过农杆菌注射法介导的瞬时表达证实了该启动子在烟草叶片中驱动GUS报告基因的表达受茉莉素的诱导．为了进-步确定该启动子中应答茉莉素信号的重要DNA区域,对该启动子进行了-系列的缺失突变,并将相关的缺失启动子片段与GUS报告基因融合．烟草叶片中GUS报告基因瞬时表达分析表明,该启动子中位于-1200bp和-800bp的碱基是该基因应答茉莉素信号的必需DNA区域,其中在-1127bp处有一个G—box元件;结合已有的研究结果,发现应答茉莉素信号的必需DNA区域不同于该基因应答ACC处理的必需DNA区域（在-562bp处存在一个ERE元件）．总之,本研究结果有助于探讨该基因应答不同胁迫信号表达的分子机制．     

木薯IPT基因的序列分析及其乙烯和茉莉酸甲酯诱导表达特性

木薯是一种重要的能源和淀粉作物,具有较强的抗逆性。为研究木薯中决定细胞分裂素合成的IPT基因如何被逆境信号调控,从木薯基因组数据库中获得了两个Me IPT基因,序列分析显示两者启动子区同时具有茉莉酸和乙烯响应元件。以木薯品种"华南八号"悬浮培养细胞为材料,利用q RT-PCR检测木薯Me IPT1和Me IPT2基因在乙烯利和茉莉酸甲酯处理后的表达模式。结果显示茉莉酸甲酯处理后两个基因显著上调,但乙烯信号对其的影响却不尽相同。这些数据说明这两个Me IPT基因可被乙烯和茉莉酸信号调控,并推测其在进一步影响细胞分裂素的生物合成中可能受整合后的不同激素信号调控。     

龙眼DCL家族基因的启动子分析及时空表达

为了解龙眼DCL基因的功能,该试验对龙眼基因组数据提取的DlDCL1、DlDCL2、DlDCL3和DlDCL4基因序列进行启动子顺式作用元件及其受miRNA调控的分析;并以龙眼胚性愈伤组织为材料,研究了DlDCLs不同基因成员在非生物胁迫和外源激素处理下的表达情况。结果显示:(1)龙眼DCL基因启动子中除了TATA和CAAT外,还具有大量的光反应元件、激素应答元件、胁迫响应元件、组织特异性调控元件及植物生长发育相关的顺式调控元件,提示龙眼DCL基因启动子转录活性可能受到光、激素信号及逆境胁迫因素的诱导。(2)对调控龙眼DCL基因的miRNA进行筛选,结果显示DlDCL1受miR162和miR1024调控,DlDCL4受miR390和miR396调控。(3)实时荧光定量PCR显示,在一定浓度范围内,外源激素GA3、ABA和ETH均能下调DlDCLs基因的表达,而高浓度ETH处理则显著上调DlDCLs的表达。(4)高浓度蔗糖(6%)处理时DlDCL2、DlDCL3和DlDCL4显著上调表达,而低浓度(0.1%)处理时DlDCL1显著上调表达;不同温度处理下,DlDCL1在34℃时显著上升,DlDCL3随着温度的提高相对表达量逐渐减低;而DlDCL2和DlDCL4表达量差异不明显;NaCl胁迫处理下,DlDCLs在1h处理时表达量下调,但在其他不同时间点则上调表达。研究表明,龙眼DCL基因在外源激素及非生物胁迫处理下,并非是简单的一对一响应,而是存在较为复杂的响应机制。     

胡杨PeNAC121基因启动子的分离鉴定和胁迫应答模式分析

为了探究NAC转录因子家族成员在胡杨（Populus euphratica）逆境胁迫中的响应和调控机制,利用PCR技术从胡杨中克隆了PeNAC121基因的启动子序列,并采用生物信息学工具对该启动子的结构特征进行了分析,最后利用该启动子驱动GUS报告基因在三倍体毛白杨（Populus tomentosa）中表达,并对获得的转基因植株采用不同胁迫处理后进行了GUS染色和酶活性定量分析。结果表明,克隆获得的PeNAC121基因的启动子长度为1 997 bp（起始密码子ATG上游）,启动序列中除了含有大量的光响应元件,还含有多个与非生物逆境胁迫和激素响应相关的元件,如低温响应元件LTR、干旱响应元件MBS、防卫和胁迫响应元件TC-rich repeats、脱落酸（ABA）响应元件、以及赤霉素（GA）响应元件等。基因的组织表达模式检测结果显示,PeNAC121基因主要在茎中表达,在根和叶中的表达较少。GUS组织化学染色和酶活性检测结果表明,胡杨PeNAC121启动子显著受到NaCl、甘露醇、ABA和4 ℃低温的诱导表达。由上述结果推测PeNAC121基因与胡杨的逆境胁迫应答密切相关,表明该基因的启动子是一个能够应答多种逆境胁迫的诱导型启动子。本研究为阐明PeNAC121基因在胡杨逆境响应和调控中的作用机制提供理论参考。     

miR398在植物逆境胁迫应答中的作用

MicroRNA (miRNA)是一类新型的调控基因表达的小分子RNA, 它作为基因表达的负调控因子, 在转录后水平调节靶基因的表达。miRNA参与调控植物的生长发育, 并在多种非生物与生物胁迫响应中发挥重要作用。miR398是第一个被报道的受氧化胁迫负调控的miRNA。它通过负调控其靶基因Cu/Zn过氧化物歧化酶(Cu/Zn-superoxide dismutase, CSD)的表达, 在多种逆境胁迫响应中扮演重要角色, 如调节铜代谢平衡, 应答重金属、蔗糖、臭氧等非生物胁迫, 以及参与应答生物胁迫等。文章综述了miR398在多种逆境胁迫响应中重要的调节作用及miR398自身的转录调控。     

水曲柳FmJAZ1基因在非生物胁迫中的响应模式和激素诱导表达分析

克隆FmJAZ1基因,明确其在低温和NaCl胁迫中的响应模式和激素诱导下的转录表达特性。通过基因克隆的方法得到水曲柳中的FmJAZ1基因,利用生物信息学软件对所得到的序列进行分析并构建系统进化树,对水曲柳FmJAZ1基因进行了时空表达特异性的分析,对根、茎、叶、芽、雄花、雌花、种子等7个部位以及在5-9月5个月份分别取样,对水曲柳进行低温(4℃)和盐胁迫(200 mmol/L NaCl)2种非生物胁迫处理以及脱落酸(ABA)、赤霉素(GA3)、生长素(IAA)、茉莉酸(JA)、水杨酸(SA)等激素信号诱导处理,然后对试验材料进行荧光定量分析。克隆出全长为684 bp的核苷酸序列。生物信息学软件分析得到JZA1基因具有完整的开放阅读框,编码227个氨基酸,JAZ1蛋白不含有信号肽,不属于跨膜蛋白,为不稳定亲水性蛋白。时空表达结果显示,FmJAZ1基因在茎中表达量最高,且在8月份表达量最高;非生物胁迫结果表明低温处理后FmJAZ1在6h、24h表达量较高;而NaCl处理后,在24 h表达量较高,且该基因响应低温胁迫较NaCl胁迫迅速;激素信号诱导结果显示,处理后不同时间,基因表达量变化较为明显,其中GA3处理后3h最为明显,为对照组的77.3倍,分析了FmJAZ1基因在低温、NaCl胁迫和激素诱导下的表达模式。FmJAZ1基因充分响应了逆境胁迫和激素信号诱导,通过蛋白和基因层面对逆境进行响应,JAZ蛋白在其中起到了桥梁的作用,并扮演了重要的角色。     

Characterization of SSEA-1 glycolipids from the brain of a patient with fucosidosis

Neutral glycolipids from the brain of a patient with Fucosidosis were analyzed and two complex glycolipids containing five and eight sugars were isolated from the cortical grey matter. These two glycolipids reacted with antibodies recognizing the SSEA-1 [Le^x(X)] carbohydrate determinant. SSEA-1 glycolipids are normally expressed in human embryonic brain but are found in only small amounts in postnatal human brain. The accumulation of the two SSEA-1 glycolipids in Fucosidosis brain thus represents a defect which affects the normal developmentally regulated decrease in postnatal, expression of these glycolipids, and may be a contributing factor in the abnormal brain development associated with the disease. Chemical characterization of the two isolated glycolipids by gas chromatographic and mass spectrometric analyses has identified the two glycolipids as lacto-N-fucopentaosylceramide (III) and difucosyl-neolactonorhexaosylceramide.Abbreviations DCl direct chemical ionization - FAB tastatiom bombardment - GC gas chromatography - GSLs glycosphingolipids - MS mass spectrometry - SSEA-1 stage specific embryonic antigen-1 - TLC thin layer chromatographys     

Elucidation of Structure of Lipid A from the Marine Gram-Negative Bacterium Pseudoalteromonas haloplanktis ATCC 14393T

The chemical structure of lipid A, from the marine -proteobacterium Pseudoalteromonas haloplanktis 14393, a main product of lipopolysaccharide hydrolysis (1% AcOH), was determined using chemical methods and NMR spectroscopy. The lipid A was shown to be -1,6-glucosaminobiose 1,4-diphosphate acylated with two (R)-3-hydroxyalkanoic acid residues at C3 and C3 and amidated with one (R)-3-hydroxydodecanoyl and one (R)-3-dodecanoyloxydodecanoyl residue at N2 and N2, respectively.     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.     

Systematic significance of mature embryo of bamboos

The mature embryo of seven species belonging to five genera of Indian bamboos is described. In all these the basic pattern of embryo organisation is same: the scutellar and coleoptilar bundles are not separated by an internode, the epiblast is absent, the lower portion of the scutellum and the coleorhiza are separated by a cleft and the margins of embryonic leaves overlap. The features unique to fleshy fruited bamboos are: presence of a massive scutellum, the juxtaposition of plumule and radicle and the occurrence of a bud in the axil of the coleoptile. The fleshy fruit bearing bamboos should be classified into one group, the tribeMelocanneae. Evidence is provided to recognise additional groups in the subfamilyBambusoideae.     

Identification of novel oocyte and granulosa cell markers

Here we present novel gene expression patterns in the ovary as part of an ongoing assessment of published micro-array data from mouse oocytes and embryos. We present the expression patterns of 13 genes that had been determined by micro-array to be expressed in the mature egg, but not during subsequent preimplantation development. In-situ hybridization of sectioned ovaries revealed that these genes were expressed in one of two distinct patterns: (1) oocyte-specific or (2) expressed in both the oocyte and surrounding granulosa cells. Despite the fact that micro-array data demonstrated expression in the egg, several of these genes are expressed at low levels in the oocyte, but strongly expressed in granulosa cells. Eleven of these genes have no reported function or expression during oogenesis, indicating that this approach is a necessary step towards functional annotation of the genome. Also of note is that while some of these gene products have been well characterized in other tissues and cell types, others are relatively unstudied in the literature. Our results provide novel gene expression information that may provide insights into the molecular mechanisms of follicular recruitment, oocyte maturation and ovulation and will direct further experimentation into the role these genes play during oogenesis.     

N-andO-glycosylation of glycoprotein C synthesized by Herpes simplex virus type 1-infected ricin resistant cells

The progeny of Herpes simplex virus type 1 (HSV-1) grown in ricin-resistant 14 cells (Ric^R14) lackingN-acetylglucosaminyltransferase I was released in the extracellular medium at a very low rate. By using a monoclonal antibody immobilized on Sepharose we purified from HSV-1-infected Ric^R14 cells a viral glycoprotein (gC), which carries bothN-andO-linked oligosaccharides. Glycopeptides obtained from [³H]mannoselabeled gC by Pronase digestion were entirely susceptible to endo--N-acetylglucosaminidase H, and the major oligosaccharide released was Man₄GlcNAc. The accumulation of this high-mannose species was related to the enzymic defect of the host cells and to the long retention of the viral glycoprotein within the cells. The extent ofO-glycosylation evaluated in [¹⁴C]glucosamine-labeled gC from Ric^R14 cells as compared to that of gC from wild type cells did not appear to be significantly modified.Abbreviations Con A concanavalin A - BHK cells baby hamster kidney cells - HSV Herpes simplex virus     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Genetics of esterase isoenzymes in Malus

Summary Three main zones of esterase activity (EST-I, EST-III, EST-IV) identified in leaf extracts of cultivated apple and Malus species were determined by the genes EST-1, EST-3 and EST-4, respectively. In addition to earlier reported alleles of EST-1 (a, b) three further bands c, d and f were identified in the EST-I zone of which c was found to be determined by an allele, c. Two alleles, a, b, and a null allele were found for both the genes EST-3 and EST-4. Differences in allelic frequency were observed between cultivars, rootstocks and Malus species. Allele EST-1a was rare amongst the rootstocks. The examination of Malus species and derivatives showed a geographical relationship. Allele EST-1c was confined to species of Asian origin, and EST-1d was confined to American species.