中间锦鸡儿CiNAC1基因促进转基因拟南芥叶片的衰老

NAC转录因子家族是植物特有的、最大的转录因子家族之一,参与植物生物胁迫和非生物胁迫应答、激素信号转导、植物次生生长、细胞分裂和植物衰老等多种过程,在植物生长发育过程中起着重要的作用。以中间锦鸡儿Ci NAC1基因的过表达拟南芥纯合体株系为材料,以野生型为对照,对Ci NAC1基因功能进行分析。结果发现,乙烯处理后,Ci NAC1基因过表达株系与野生型拟南芥相比,叶片衰老提前、叶绿素含量降低、离子渗透率升高。实时荧光定量PCR检测发现,乙烯处理后Ci NAC1基因过表达株系中与叶绿素降解相关的基因SGR1、SGR2、PPH,以及与衰老相关的基因SAG13、SAG29、ORE1、SINA1、VNI2和乙烯信号途径中的重要转录因子EIN3的表达量均明显高于野生型拟南芥。表明Ci NAC1基因在乙烯诱导的叶片衰老过程中发挥重要作用。     

转CkLEA4基因拟南芥种子萌发期的抗逆性分析

该研究在实验室前期研究的基础上,将受脱水、盐胁迫和ABA诱导的柠条锦鸡儿CkLEA4基因转入野生型拟南芥,并利用实时荧光定量PCR从8株纯合体中筛选出3个表达量不同的株系,比较野生型和转CkLEA4基因过表达拟南芥种子在不同胁迫处理下的萌发率,以探讨CkLEA4基因在植物抵抗逆境胁迫中的功能。结果发现:(1)在不同浓度NaCl、甘露醇及ABA处理下,转CkLEA4基因过表达拟南芥种子的萌发率均高于野生型,随着NaCl、甘露醇及ABA浓度增加,各株系萌发率均降低,但野生型的萌发率下降幅度均高于3个过表达株系,并且在200mmol/L NaCl和400mmol/L甘露醇处理下,过表达株系子叶绿化率均显著高于野生型。(2)在低浓度ABA处理下,CkLEA4过表达植株子叶的绿化率也高于野生型。研究表明,柠条锦鸡儿CkLEA4基因提高了拟南芥种子萌发阶段对盐、ABA及渗透胁迫的耐受性。     

转ZjAPX基因拟南芥对NaCl、干旱胁迫的耐性研究

旨在探讨枣树抗坏血酸过氧化物酶基因ZjAPX在植物渗透胁迫中的作用。将ZjAPX基因转入到模式植物拟南芥,以野生型(WT)、转ZjAPX拟南芥株系T2为试材,进行不同浓度NaCl胁迫和干旱胁迫。结果表明,转基因株系的种子萌发、植株生长均优于野生型株系;荧光定量PCR检测转基因拟南芥植株在干旱和盐胁迫处理10 d后目的基因ZjAPX的表达量显著高于野生拟南芥,表明ZjAPX的高表达明显提高了植株的抗旱和耐盐性。     

玉米分子伴侣基因ZmBiP1的表达模式和功能研究

以抗旱玉米自交系旱21为材料,克隆获得玉米分子伴侣基因Zm Bi P1,通过荧光定量PCR技术研究Zm Bi P1基因的表达模式,并将Zm Bi P1基因转入拟南芥中研究其功能。荧光定量PCR结果表明,Zm Bi P1基因在玉米自交系旱21的不同组织器官中均有表达,且在子房中表达量最高。Zm Bi P1基因受甘露醇胁迫诱导上调表达,受盐胁迫诱导下调表达。转基因拟南芥功能验证结果表明,过表达Zm Bi P1基因导致拟南芥在种子萌发时期对盐和甘露醇胁迫的耐受能力减弱。这些结果表明Zm Bi P1基因可能是逆境反应信号传递途径的负调节因子。     

VHA-c2&c4双基因沉默株系拟南芥对NaCl与ABA的响应

研究液泡H~+-ATPase c亚基基因(VHA-c2和VHA-c4)在植物生长发育及响应非生物胁迫过程中的功能,构建拟南芥VHA-c2c4的RNAi表达载体,通过浸花法转化野生型拟南芥。利用卡那霉素筛选纯合体株系,进行半定量PCR鉴定,并对其进行NaCl和ABA胁迫处理。获得7个T_2代双沉默基因纯合体株系,其VHA-c2和VHA-c4的mRNA表达水平均低于野生型。挑选沉默效率较高的3个株系进行NaCl和ABA处理,在NaCl处理下,它们的主根相对伸长量和种子萌发率均小于对照,表明VHAc2c4沉默株系对NaCl胁迫作用敏感。ABA处理下,子叶的展开程度和主根相对伸长量均比对照高,表明VHA-c2c4沉默株系对ABA抑制作用不敏感。NMT试验表明ABA促进了双沉默基因纯合体株系的H+内流能力。推测VHA-c2和VHA-c4的转录水平影响了植物对盐胁迫和ABA介导的信号转导途径的响应。     

盐胁迫诱导盐穗木Rev1、Rev3基因的表达分析

根据盐穗木盐胁迫下响应的转录组测序结果,参考盐穗木HcRev1、HcRev3基因的ESTs序列设计荧光定量PCR特异性引物,建立检测盐穗木Revs基因相对表达量的荧光定量PCR方法,分析Rev1和Rev3基因在盐穗木不同浓度盐胁迫处理不同时间的转录水平。结果表明,HcRev1、HcRev3基因具有相似的表达模式,在100 mmol·L^-1 NaCl低盐胁迫下表达稳定,在300、500、700 mmol·L^-1 NaCl胁迫下,随胁迫浓度增高、胁迫时间延长,表达量升高。其中HcRev1在700 mmol·L^-1 NaCl胁迫14 d后达到峰值,是对照组的4.63倍。HcRev3基因在300 mmol·L^-1 NaCl胁迫14 d时,表达量迅速升高,是对照组的15.55倍,表达差异极显著。研究结果说明HcRev1、HcRev3基因都受盐胁迫诱导表达,提示HcRev1、HcRev3基因虽然表达量存在差异,但在盐胁迫过程中参与了DNA损伤修复。研究有助于阐明Rev1、Rev3基因在DNA损伤修复和植物耐盐性间的调控功能作用。     

过表达SmERF1提高了丹参耐盐性

ERF(ethylene-responsive factor)蛋白是植物中一大类转录因子家族,在植物的生长发育及逆境胁迫中发挥着重要作用.为研究丹参中新的转录因子SmERF1的功能,本研究对其编码序列、表达模式进行分析,并在丹参中进行过表达,检测过表达后转基因植株耐盐性、次生代谢物和激素的变化.研究结果表明,SmERF1含有一个AP2结构域,是典型的ERF转录因子.SmERF1受到MeJA和酵母诱导子(YE)的诱导表达.在盐胁迫下,过表达SmERF1的丹参株系中脯氨酸含量、SOD和POD活性较野生型丹参植株高,MDA含量降低,说明转基因株系的抗盐特性增强.而且过表达SmERF1的丹参株系中ABA含量升高,GA含量降低;过表达SmERF1对丹参酮、丹酚酸等代谢物的合成调控效果不强.综合以上结果,表明SmERF1可能通过调节ABA的合成,提高了丹参耐盐性.     

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。     

小拟南芥ApZFP基因异源超表达促进拟南芥开花并提高耐逆性

锌指蛋白(ZFP)是一类重要的转录因子, 广泛参与植物的生长发育和非生物胁迫应答。新疆小拟南芥(Arabidopsispumila)又名无苞芥, 是十字花科短命植物, 具有高光效、繁殖力强和适应干旱等生物学特征, 而且比模式植物拟南芥(A.thaliana)更耐高盐胁迫。将前期克隆的小拟南芥锌指蛋白基因ApZFP通过花滴法转化到哥伦比亚生态型拟南芥(Col-0)中,获得了独立表达的转基因株系。表型观察发现, 过量表达ApZFP基因可促使拟南芥在长短日照下均提前开花。实时荧光定量PCR结果显示, 转基因拟南芥株系中, 光周期途径中的CO基因和年龄途径中的SPL基因表达上调; 春化、环境温度和自主途径中的FLC基因表达下调; 编码成花素的基因FT及下游开花相关基因AP1和LFY的表达量均升高。进一步通过盐、干旱和ABA胁迫处理ApZFP转基因株系的种子和幼苗, 发现在胁迫处理下, 与对照相比, 转基因拟南芥种子萌发率较高, 幼苗主根较长。因此推测, ApZFP在植物发育过程中具有多种功能, 可能既参与植物的开花转变过程, 又同其它植物的锌指蛋白基因一样, 参与植物的耐逆过程。     

盐穗木miRNA417的克隆及对种子萌发和幼苗成活率的影响

MicroRNA (miRNA)是植物重要的基因表达调控因子,miR417的表达受盐胁迫的调节,高盐胁迫时,拟南芥miR417的表达能够抑制种子的萌发和幼苗成活。本研究通过分析miRbase数据库中已知植物miRNA417的序列,利用PCR技术成功克隆获得了盐生植物盐穗木的miR417（HcmiR417）的前体序列,将其构建至植物表达载体pCAMBIA1301上,通过花絮浸染法对拟南芥进行遗传转化。结果表明,在150 mmol·L^-1 NaCl的胁迫下,分别过表达HcmiR417和过表达拟南芥miRNA417（AtmiR417）的转基因拟南芥种子的萌发率和幼苗存活率均较野生型低,但两种转基因拟南芥株系之间没有差异。初步验证了盐生植物HcmiR417在种子萌发和幼苗成活率方面也具有负调控作用,盐生植物盐穗木和拟南芥植物miRNA在功能没有显示出差异。     

Preparation of hair for measurement of elements by inductively coupled plasma-mass spectrometry (ICP-MS)

The preparation of hair for the determination of elements is a critical component of the analysis procedure. Open-beaker, closedvessel microwave, and flowthrough microwave digestion are methods that have been used for sample preparation and are discussed. A new digestion method for use with inductively coupled plasma-mass spectrometry (ICP-MS) has been developed. The method uses 0.2 g of hair and 3 mL of concentrated nitric acid in an atmospheric pressurelow-temperature microwave digestion (APLTMD) system. This preparation method is useful in handling a large numbers of samples per day and may be adapted to hair sample weights ranging from 0.08 to 0.3 g. After digestion, samples are analyzed by ICP-MS to determine the concentration of Li, Be, B, Na, Mg, Al, P, S, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ge, As, Se, Rb, Sr, Zr, Mo, Pd, Ag, Cd, Sn, Sb, I, Cs, Ba, Pt, Au, Hg, Tl, Pb, Bi, Th, and U. Benefits of the APLTMD include reduced contamination and sample handling, and increased precision, reliability, and sample throughput.     

A phylogenetic analysis of 100+ genera and 50+ families of euasterids based on morphological and molecular data with notes on possible higher level morphological synapomorphies

 A data matrix of 143 morphological and chemical characters for 142 genera of euasterids according to the APG system was compiled and complemented with rbcL and ndhF sequences for most of the genera. The data were subjected to parsimony analysis and support was assessed by bootstrapping. Strict consensus trees from analyses of morphology alone and morphology + rbcL + ndhF are presented. The morphological data recover several groups supported by molecular data but at the level of orders and above relationships are only superficially in agreement with molecular studies. The analyses provide support for monophyly of Gentianales, Aquifoliales, Apiales, Asterales, and Dipsacales. All data indicate that Adoxaceae are closely related to Dipsacales and hence they should be included in that order. The trees were used to assess some possible morphological synapomorphies for euasterids I and II and for the orders of the APG system. Euasterids I are generally characterised by opposite leaves, entire leaf margins, hypogynous flowers, “early sympetaly” with a ring-shaped corolla primordium, fusion of stamen filaments with the corolla tube, and capsular fruits. Euasterids II often have alternate leaves, serrate-dentate leaf margins, epigynous flowers, “late sympetaly” with distinct petal primordia, free stamen filaments, and indehiscent fruits. It is unclear which of these characters represent synapomorphies and symplesiomorphies for the two groups, respectively, and there are numerous expections to be interpreted as reversals and parallelisms. Received August 28, 2000 Accepted August 7, 2001     

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein^1,2. The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale^1-4.Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).     

Automated High-throughput Behavioral Analyses in Zebrafish Larvae

We have created a novel high-throughput imaging system for the analysis of behavior in 7-day-old zebrafish larvae in multi-lane plates. This system measures spontaneous behaviors and the response to an aversive stimulus, which is shown to the larvae via a PowerPoint presentation. The recorded images are analyzed with an ImageJ macro, which automatically splits the color channels, subtracts the background, and applies a threshold to identify individual larvae placement in the lanes. We can then import the coordinates into an Excel sheet to quantify swim speed, preference for edge or side of the lane, resting behavior, thigmotaxis, distance between larvae, and avoidance behavior. Subtle changes in behavior are easily detected using our system, making it useful for behavioral analyses after exposure to environmental toxicants or pharmaceuticals.     

外泌体在白血病中的重要调控作用

目前,白血病复发是患者死亡的主要原因之一。肿瘤细胞和微环境的相互作用,以及隐匿在骨髓中的肿瘤干细胞,促进了白血病的复发和向淋巴组织的转移,因此白血病的治疗、转移和复发问题受到广泛关注。外泌体是由绝大多数细胞分泌的双层脂质膜囊泡,可以调控细胞间的交流和信息传递。在白血病细胞、基质细胞和内皮细胞之间的相互联系中都涉及到外泌体,白血病细胞来源的外泌体存在于白血病患者的血浆中,能把其携带的白血病相关抗原及微小RNA呈递给靶细胞,促进白血病肿瘤细胞的增殖,有助于肿瘤细胞实现免疫逃避,保护白血病细胞抵抗化疗药物导致的细胞毒性作用,促进血管生成及肿瘤细胞的迁移。因此,外泌体与白血病的转移、治疗及预后密切相关,可以用来检测和监测白血病的进展。本文综述了外泌体的来源、形成与分泌机制,以及外泌体在白血病发生前、发展中、预后和免疫治疗中所扮演的重要角色。     

Assessing burrowing, nest construction, and hoarding in mice

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening of possible treatments for AD currently largely focuses on cognitive testing, which frequently demands expensive equipment and lots of experimenter time. However, human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which seems to be largely non-episodic. Therefore the present ways of screening for new AD treatments for AD in rodents are intrinsically unlikely to succeed. A new approach to preclinical screening would be to characterise the ADL of mice. Fortuitously, several such assays have recently been developed at Oxford, and here the three most sensitive and well-characterised are presented. Burrowing was first developed in Oxford. It evolved from a need to develop a mouse hoarding paradigm. Most published rodent hoarding paradigms required a distant food source to be linked to the home cage by a connecting passage. This would involve modifying the home cage as well as making a mouse-proof connecting passage and food source. So it was considered whether it would be possible to put the food source inside the cage. It was found that if a container was placed on the floor it was emptied by the next morning., The food pellets were, however, simply deposited in a heap at the container entrance, rather than placed in a discrete place away from the container, as might be expected if the mice were truly hoarding them. Close inspection showed that the mice were performing digging ("burrowing") movements, not carrying the pellets in their mouths to a selected place as they would if truly hoarding them. Food pellets are not an essential substrate for burrowing; mice will empty tubes filled with sand, gravel, even soiled bedding from their own cage. Moreover, they will empty a full tube even if an empty one is placed next to it. Several nesting protocols exist in the literature. The present Oxford one simplifies the procedure and has a well-defined scoring system for nest quality. A hoarding paradigm was later developed in which the mice, rather than hoarding back to the real home cage, were adapted to living in the "home base" of a hoarding apparatus. This home base was connected to a tube made of wire mesh, the distal end of which contained the food source. This arrangement proved to yield good hoarding behaviour, as long as the mice were adapted to living in the "home base" during the day and only allowed to enter the hoarding tube at night.     

日本北海道虻类雌性外生殖器形态(双翅目：虻科)(英文)

【目的】利用日本北海道虻类评估和验证外生殖器在分类学上的意义。【方法】将虻类成虫标本浸渍在生理盐水中并置于双目显微镜下通过针和镊子在培养皿中进行解剖并绘图,观察第9背板、第10背板、尾叶、第8腹板、受精囊、受精囊管及生殖叉器的形态特征。【结果】在日本北海道共记录了虻科(Tabanidae) 3亚科7属38种。我们观察并描述了3亚科其中的6属24种的雌性外生殖器的主要特征。亚科之间存在明显差异;然而在一般情况下属之间很难建立一种方法来确定共同点;种之间只有在斑虻属Chrysops中有相似之处,其他属中则比较多样化。因此,亚科鉴定根据第9背板、第8腹板及受精囊足以进行区分,属及种鉴定需要结合第9背板、第10背板、尾叶、第8腹板、受精囊、受精囊管及生殖叉器各自的特征组合在一起才能区分开来。我们也制作了虻类外生殖器的检索表。【结论】和许多其他昆虫一样,外生殖器是虻科的重要分类特征,对于促进分类学和系统学的发展具有重要意义。本研究首次对分布在日本北海道的虻科雌性外生殖器进行了系统研究。     

The Rumen Ciliate Fauna of Domestic Sheep (Ovis ammon aires) from the Turkish Republic of Northern Cyprus

ABSTRACT. Concentration and composition of ciliate protozoa in the families Ophryoscolecidae and Isotrichidae were determined in rumen contents of domestic sheep ( Ovis ammon aries ) from Cyprus. A total of five genera of Ophryoscolecidae were identified, Metadinium, Enoploplastron, Polyplastron, Epidinium , and Ophryoscolex , which included six species: Metadinium affine, Enoploplastron triloricatum, Polyplastron multivesiculatum, Epidinium ecaudatum, Epidinium graini, and Ophryoscolex purkynjei. Eight separate forms of Epidinium were identified ( E. ecaudatum f. ecaudatum, E, e. f. caudatum, E. e. f. bicaudatum, E. e. f. tricaudatum, E. e. f. quadricaudatum, E. graini f. graini, E. g. f. caudatricoronatum , and E. g. f. caudaquadricoronatum ), along with five forms of Ophryoscolex purkynjei (O. p. f. purkynjei, O. p. f. bifidobicinctus, O. p. f. bifidoquadricinctus, O. p. f. bicoronatus, O. p. f. tricoronatus , and O. p. f. quadricoronatus). Three species of Isotrichidae were observed, Isotricha intestinalis, I. prostoma , and Dasytricha ruminantium. This study reports new host records for three forms of Epidinium graini and Ophryoscolex purkynjei f. bifidobicinctus. The rumen fauna in the family Ophryoscolecidae from Cypriote domestic sheep appear to have limited diversity compared to those from Turkish and Far Eastern (Chinese/Japanese) sheep, while they are more diverse than those found in Western European (Scottish) and North American (Canadian/Alaskan) sheep.     

中国辽秃蝗属的分类研究及二新种记述（直翅目， 蝗总科， 斑腿蝗科， 秃蝗亚科）（英文）

记述了我国辽秃蝗属Liaopodisma 3个种, 包括2新种： 台中辽秃蝗Liaopodisma taichungensis sp. nov.和台湾辽秃蝗Liaopodisma taiwanensis sp. nov.。台中辽秃蝗L. taichungensis sp. nov.同千山辽秃蝗L. qianshanensis Zheng, 1990近似, 不同之处为黑色眼后带不明显, 前胸背板中隆线几乎不见, 雄性前翅长为宽的1.8倍, 雄性中胸腹板中隔向后渐宽大, 雄性后足胫节外侧具10刺。台湾辽秃蝗L. taiwanensis sp. nov.的后足股节下侧红色, 前翅中部不加宽且前、 后缘几乎平行, 可与本属其余2已知种区别。并附辽秃蝗属种检索表。     

Unraveling regulation and new components of human P-bodies through a protein interaction framework and experimental validation

The cellular factors involved in mRNA degradation and translation repression can aggregate into cytoplasmic domains known as GW bodies or mRNA processing bodies (P-bodies). However, current understanding of P-bodies, especially the regulatory aspect, remains relatively fragmentary. To provide a framework for studying the mechanisms and regulation of P-body formation, maintenance, and disassembly, we compiled a list of P-body proteins found in various species and further grouped both reported and predicted human P-body proteins according to their functions. By analyzing protein-protein interactions of human P-body components, we found that many P-body proteins form complex interaction networks with each other and with other cellular proteins that are not recognized as P-body components. The observation suggests that these other cellular proteins may play important roles in regulating P-body dynamics and functions. We further used siRNA-mediated gene knockdown and immunofluorescence microscopy to demonstrate the validity of our in silico analyses. Our combined approach identifies new P-body components and suggests that protein ubiquitination and protein phosphorylation involving 14-3-3 proteins may play critical roles for post-translational modifications of P-body components in regulating P-body dynamics. Our analyses provide not only a global view of human P-body components and their physical interactions but also a wealth of hypotheses to help guide future research on the regulation and function of human P-bodies.