转录因子cpcR同源基因cpcR-c1和cpcR-t的功能鉴定

苏云金芽胞杆菌（Bacillus thuringiensis,Bt） LM1212菌株与典型的Bt菌株表型不同,可分化形成芽胞、形成细胞和晶体产生细胞。在LM1212菌株中,转录因子CpcR不仅参与了细胞分化过程,而且能够激活晶体蛋白基因cry35-like的启动子(P₃₅)。【目的】筛选cpcR同源基因,验证其生物学功能。【方法】本研究克隆了2个cpcR同源基因,来源于蜡样芽胞杆菌的cpcR-c1和来源于东洋芽胞杆菌的cpcR-t,将cpcR及其同源基因分别构建在pHT304-P₃₅-gfp、pHT304-P₃₅-lacZ报告载体上,获得的重组质粒转入无cpcR基因且无晶体蛋白基因的Bt HD73^–菌株中。利用激光共聚焦显微镜观察重组菌HD^–(cpcR-c1-P₃₅-gfp)和HD^–(cpcR-t-P₃₅-gfp)的细胞表型并进行芽胞计数实验。测定HD^–(cpcR-c1-P_35<...     

元宝枫花芽分化及花开放过程研究

为探明元宝枫花性别、交配系统及花芽分化过程,通过石蜡切片和连续解剖观察,对元宝枫花开放及花芽分化过程进行了研究。结果表明：(1)元宝枫花性别可分为雌能花和雄能花两种,雌能花花药不开裂,只表现雌性功能,而雄能花分Ⅰ型和Ⅱ型两种,花药均可产生成熟花粉并开裂散粉,只表现雄性功能。(2)元宝枫是较为少见的二重雌雄异型异熟树种,且交配系统有雄先型和雌先型两种;雄先型小花开放顺序为：雄能花→雌能花→雄能花,雌先型小花开放顺序为雌能花→雄能花→雄能花。(3)元宝枫花芽的形态分化期开始于7月上旬,花序和小花分化期为8月上旬,雄蕊和雌蕊分化开始于8月下旬,雌配子体发育晚于雄配子体。(4)元宝枫花性别分化的关键期为第二年的3月下旬至4月上旬。     

化龙山七筋姑种内二倍体与四倍体表型分化分析

七筋姑(Clintonia udensis Trautv. et Mey.)属百合科(Liliaceae)七筋姑属(Clintonia Raf.),多年生草本,具有二倍体(2n=14)和四倍体(4n=28)两种倍性。在陕西化龙山地区,二倍体主要分布在南坡海拔2 450 m处,四倍体主要生长在北坡海拔1 900 m左右,成为研究种内多倍体分化的理想材料。该研究从七筋姑营养和繁殖系统出发,研究其不同倍性的表型分化,揭示不同倍性的生态适应特征,为阐明七筋姑种内多倍体的演化提供线索。研究表明：(1)在根、茎、叶、花、果实、种子的11个性状中,二倍体的果实体积性状最为稳定(CV=0.02),叶长性状遗传多样性最高(CV=0.85);四倍体中果实体积性状也最为稳定(CV=0.06),而花数量性状多样性最高(CV=0.42)。(2)四倍体的果实体积平均值明显高于二倍体,但种子数量平均值显著少于二倍体,果实体积和种子体积性状在不同倍性间的分化占比最高(V_st=0.69)。(3)四倍体营养器官表型性状的遗传变异丰富度低于二倍体,其平均变异系数(CV=0.16)小于二倍体的变异系数(CV=0.44);在繁殖系统表型性状中,四倍体的遗传变异丰富度高于二倍体,平均变异系数(CV=0.30)大于二倍体(CV=0.26)。(4)显著性分析表明,二倍体与四倍体表型性状差异显著(P<0.05),而繁殖系统性状在不同倍性间无显著差异(P>0.05);PCA分析同样显示二倍体与四倍体间存在明显差异。四倍体显著的表型差异是对低海拔环境长期适应的结果。     

Naegleria fowleri: functional expression of the Nfa1 protein in transfected Naegleria gruberi by promoter modification

To establish a transient transfection system in a Naegleria, we constructed three nfa1-pEGFP-N1 vectors by the promoter replacement and insertion of a nfa1 gene and transfected the DNAs into Naegleria gruberi using a lipid reagent. The transfection efficiency and usefulness of the three modified vectors were estimated by identifying the expressions of the EGFP and Nfa1 protein from N. gruberi. After transfection, the Nfa1 protein was functionally expressed on pseudopodia of N. gruberi. The strong GFP fluorescence was observed in N. gruberi transfected with the actin-nfa1-pEGFP-N1 vector, of which the CMV promoter region in the expression vector was replaced with the actin 5' UTR region. Additionally, when transgenic N. gruberi trophozoites were co-cultured with CHO target cells, the Nfa1 protein was also located on cytoplasm and pseudopodia, especially on a food cup that was formed in contact with target cells as it shown in pathogenic N. fowleri.     

杠柳花芽分化过程的形态解剖学特征

杠柳是萝藦科杠柳属蔓性灌木,是庭院、假山、林缘地带垂直绿化的地被植物。为了探究杠柳在吉林延边地区的生长节律以及花芽分化和开花动态,该研究以吉林向海湿地保护区采集的杠柳种子为材料,播种在延边大学农学院试验田,连续追踪5年进行观测,并采用田间观察和常规石蜡切片法对杠柳的生长节律和花芽分化各阶段形态解剖特征进行研究。结果表明：(1)在吉林延边地区,杠柳种子萌发后的第一年和第二年主要进行营养生长,第三年能形成花芽,进入生殖生长期。(2)在延边地区杠柳每年开花最早在5月末,花蕾期在5月末至6月初,始花期在6月初至6月中旬,盛花期在6月中旬至6月末,末花期在6月末至7月初,结果结实期在7月初到10月中旬。(3)杠柳于5月下旬开始进入花芽形态分化阶段,其花芽分化过程包括：未分化期、分化初期、花萼原基分化期、花瓣原基分化期、雄蕊原基分化期、雌蕊原基分化期和副花瓣原基分化期。     

The phylogenetic placement of the Leucogastrales, including Mycolevis siccigleba (Cribbeaceae), in the Albatrellaceae using morphological and molecular data

The morphology of many hypogeous fungi converges on a homogeneous reduced form, suggesting that disparate lineages are subject to a uniform selection pressure. The primary goal of this study was to evaluate the morphology and infer the phylogeny of the Leucogastrales with Mycolevis siccigleba using a Bayesian methodology. A comprehensive morphological assessment was used for an a priori phylogenetic inference to guide the sequencing effort. All structures except spore ornamentation pointed to the Albatrellaceae as the most likely sister taxon. Polyporoletus sublividus, a close relative of Albatrellus, produces ornamented basidiospores with a similar structure to M. siccigleba basidiospores. The ITS from 30 taxa was used for the molecular phylogenetic analysis. P. sublividus was found sister to Mycolevis. Leucophleps spinispora and L. magnata formed a group sister to the Polyporoletus/Mycolevis group, whereas Leucogaster was polyphyletic with respect to the core of the Leucogastrales and sister to A. caeruleoporus. This relationship was expected as previously undescribed chlamydospores produced by members of Albatrellus had a similar morphology to the basidiospores of L. rubescens.     

栗背短脚鹎的种群分化与遗传多样性

栗背短脚鹎(Hemixos castanonotus)是我国南方山区常见的杂食性鸟类。为探明其遗传多样性及分化现状,采用线粒体Cyt b基因和7个核基因非编码区片段作为分子标记,对分布于广东、广西、海南、贵州和江西五省(自治区)的栗背短脚鹎11个地理种群进行了遗传分化及遗传多样性研究。基于所获得的Cyt b基因866 bp和7个核基因内含子序列6 808 bp进行分析。结果显示,在Cyt b基因中,共检测到37个单倍型,共享单倍型占单倍型总数的35.6%,推测这些共享单倍型可能属于祖先单倍型。分子方差分析结果显示,遗传变异主要来源于种群内部(79.77%)。Tajima’s D和Fu’s Fs中性检验分析结果均支持栗背短脚鹎种群可能曾经历过种群扩张现象。基于7个核基因内含子联合序列的贝叶斯天际线(BSP)分析,推断其种群在大约5.3 ~ 3.7百万年前(Mya)和约0.7 ~ 0.3百万年前(Mya)发生过扩张。基于Cyt b基因的贝叶斯系统发育分析,11个地理种群共分为两支,一支为海南猴猕岭地理种群,属指名亚种(H. c. castanonotus),其他10个地理种群聚为另一支,属H. c. canipennis亚种,并且后者尚未形成显著的地理结构,单倍型网络图分析也获得相似的结果。本研究所用分子数据基本支持两个亚种的分化,对于存在争议的广西南部分布的指名亚种,其分子数据与形态学亚种归属不一致,有待更深入研究。     

六角果鸢尾离体快繁技术体系的构建

为构建六角果鸢尾(Iris hexagona)的离体快繁体系,以其幼嫩根状茎为外植体,研究了培养基和植物生长调节剂对不定芽诱导、增殖和植株生根的影响。结果表明,根状茎用0.1%Hg Cl2消毒13 min的效果较佳;不定芽诱导最适培养基为MS+6-BA 1.5 mg L–1+NAA 0.5 mg L–1+蔗糖30 g L–1+琼脂7.5 g L–1,不定芽增殖的适宜培养基为MS+6-BA 0.5 mg L–1+NAA0.2 mg L–1+KT 0.3 mg L–1+蔗糖30 g L–1+琼脂7.5 g L–1;在MS+IBA 1.5 mg L–1+蔗糖30 g L–1+琼脂7.5 g L–1培养基上不定芽生根率可达100%;腐殖土和珍珠岩+泥炭土+蛭石(1∶2∶1)均可作为组培苗移栽的适宜基质,移栽成活率可达100%。     

Phylogeographic patterns in Leccinum sect. Scabra and the status of the arctic-alpine species L. rotundifoliae

We investigated inter- and intraspecific phylogenetic relationships in the ectomycorrhizal fungal genus Leccinum section Scabra. Species of this section are exclusively associated with Betula and occur throughout the Northern Hemisphere. We compared the phylogenetic relationships of arctic, alpine, boreal and temperate accessions of section Scabra based on DNA sequences of the single-copy nuclear gene Gapdh and the multiple-copy nuclear region 5.8S-ITS2. Exclusively arctic lineages were not detected in species that occur both in arctic-alpine or boreal regions, except in L. rotundifoliae that was restricted to cold climates. L. scabrum and L. holopus showed an intercontinental phylogeographic pattern, and L. variicolor showed a pattern unrelated to geographical distribution. Molecular clock estimates indicated that L. rotundifoliae is as old as other species in section Scabra. Individual gene trees suggest that interspecific hybridisation occurred several times in the evolution of section Scabra.     

Ameboflagellate transformation I. Possible initiation of the ameba-to-flagellate transformation of Naegleria gruberi (NB-1) by divalent cation influx

The hypothesis that the ameba-to-flagellate transformation (AFT) of Naegleria gruberi (NB-1) requires Ca²⁺ and/or Mg²⁺ was tested with divalent cation blockers and competitors. Several compounds were found to decrease the rate and magnitude of the AFT as well as increasing the time of enflagellation onset. The compounds include: the chloride salts of lanthanum, cobalt, and manganese; C-600; and EDTA. These results suggest that the AFT requires the influx of Ca²⁺ and/or Mg²⁺.Supported by NIH Training Grant 5-T01-N505670     

基因CfATG6和CfATG14参与调控果生刺盘孢细胞自噬和致病力

【目的】炭疽病是油茶的主要病害,由刺盘孢属的多种真菌引起,其中果生刺盘孢分布范围最广、分离率最高,是油茶炭疽病的主要致病菌。研究自噬相关蛋白CfAtg6和CfAtg14的生物学功能,为进一步揭示果生刺盘孢通过细胞自噬调控致病的分子机制,并为油茶炭疽病的防治提供理论基础。【方法】根据同源重组原理,通过聚乙二醇(polyethylene glycol, PEG)介导的方法,在果生刺盘孢中敲除基因CfATG6和CfATG14,并进一步获得回补菌株ΔCfatg6-C和ΔCfatg14-C。【结果】酵母双杂交试验结果显示,果生刺盘孢蛋白CfAtg6和CfAtg14可能存在互作关系。生物学表型测定结果表明,相较于野生型和回补菌株,突变体ΔCfatg6和ΔCfatg14均表现出营养生长速率显著减慢,附着胞形成率分别只有野生型的5%和18%;突变体ΔCfatg6和ΔCfatg14致病力均极显著减弱,造成的油茶叶片病斑面积少于野生型和回补菌株的1/3;CfATG6和CfATG14基因缺失突变体均丧失转运和降解CfAtg8蛋白的能力,并对细胞壁胁迫更敏感。突变体ΔCfatg6的分生孢子产量显著降低,仅为野生型的20%左右;氧化胁迫试验结果表明,相较于野生型和回补菌株,过氧化氢对突变体的生长抑制率升高10%左右。内质网压力胁迫试验表明,ΔCfatg14对二硫苏糖醇抑制率升高5%以上。【结论】自噬相关基因CfATG6和CfATG14参与调控了果生刺盘孢生长发育、细胞自噬和致病力。     

Retinoic Acid-Induced blr1 Expression Promotes ERK2 Activation and Cell Differentiation in HL-60 Cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G₁/G₀ growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1α,25-dihydroxyvitamin D₃ and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G₁/G₀ growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.     

A polysaccharide from Lichina pygmaea and L. confinis supports the recognition of Lichinomycetes

The lichen-forming order Lichinales, generally characterized by prototunicate asci and the development of thalli with cyanobacteria, has recently been recognized as a separate class of ascomycetes, Lichinomycetes, as a result of molecular phylogenetic studies. As alkali and water-soluble (F1SS) polysaccharides reflect phylogeny in other ascomycetes, a polysaccharide from Lichina pygmaea and L. confinis was purified and characterized to investigate whether these F1SS compounds in the Lichinomycetes were distinctive. Nuclear magnetic resonance (NMR) spectroscopy and chemical analyses revealed this as a galactomannan comprising a repeating unit consisting of an α-(1→6)-mannan backbone, mainly substituted by single α-galactofuranose residues at the O-2- or the O-2,4- positions linked to a small mannan core. With the exception of the trisubstituted mannopyranose residues previously described in polysaccharides from other lichens belonging to orders now placed in Lecanoromycetes, the structure of this galactomannan most closely resembles those found in several members of the Onygenales in Eurotiomycetes. Our polysaccharide data support molecular studies showing that Lichina species are remote from Lecanoromycetes as the galactofuranose residues are in the α-configuration. That the Lichinomycetes were part of an ancestral lichenized group can not be established from the present data because the extracted polysaccharide does not have the galactofuranose residue in the β configuration; however, the data does suggest that an ancestor of the Lichinomycetes contained a mannan and was part of an early radiation in the ascomycetes.     

罗汉果花芽分化过程中形态及其激素水平变化特征

采用石蜡切片和酶联免疫法(ELISA)对罗汉果雄性、雌性、两性花芽分化过程的形态和激素水平变化进行观测,为罗汉果开花调控和品种选育提供科学依据。结果表明:(1)罗汉果雄性、雌性、两性花的花芽分化过程均可分为花芽未分化期、花芽分化初期、花序分化期、萼片原基分化期、花瓣原基分化期、雄蕊原基分化期和雌蕊原基分化期7个阶段。雄蕊原基分化期前,3种花芽分化过程无明显差异,各时期形态特征均依次为:茎端呈圆锥状(花芽未分化期)→茎端经半球形变成扁平状(花芽分化初期)→距茎端5~7节位处分化出穗状花序(花序分化期)→小花原基周围形成5个萼片原基(萼片原基分化期)→萼片原基内侧形成5个花瓣原基(花瓣原基分化期)。雄蕊和雌蕊原基分化期,3种花芽分化过程存在明显差异,雄蕊原基内侧出现雌蕊原基后,雄花芽雄蕊原基继续发育成雄蕊,雌蕊原基停滞生长,退为一个小突起;雌花芽雌蕊原基继续发育成雌蕊,雄蕊原基生长缓慢,退化为小花丝;两性花芽雌蕊和雄蕊原基均继续发育,形成外观正常的雌蕊和雄蕊。(2)内源激素脱落酸(ABA)、赤霉素(GAs)和玉米素核苷(ZR)含量在3种花芽分化过程中变化规律相似,即ABA含量在花芽生理分化期降低,花芽形态分化期升高,而GAs和ZR含量则基本保持不变;吲哚乙酸(IAA)含量在3种花芽分化过程中变化存在明显差异,雌花芽IAA含量在花芽生理分化期升高,花芽形态分化期逐渐降低,而雄性和两性花芽的IAA含量则基本保持不变。ABA/GAs、ABA/IAA、ZR/IAA和ZR/GAs激素含量比值在3种花芽分化过程中变化规律相似,ABA/GAs在花芽生理分化期降低,花芽形态分化期升高,而BA/IAA、ZR/IAA和ZR/GAs则基本保持不变。研究认为,罗汉果花芽分化过程经历一个"两性期",高ABA含量和ABA/GAs比值有利于罗汉果花芽分化,IAA可能对罗汉果花性分化具有重要作用。     

文心兰COL基因的分离及其表达分析

CONSTANS(CO)及CONSTANS-like(COL)基因在光周期调控植物开花中起到重要的作用。该研究以文心兰(Oncidium)品种‘金辉’为材料,分离了CO同源基因OnCOL2及另外2个COL基因(OnCOL8和OnCOL9),它们分别编码326、411和291个氨基酸;生物信息分析预测它们均定位于细胞核;OnCOL2、OnCOL8有2个锌指B-box结构域和1个CCT结构域,而OnCOL9缺少B-box结构域。多序列比对及进化树分析结果表明,所有COL蛋白可划分为3组,OnCOL2与OnCOL8、OnCOL9分到了2个不同组中。OnCOL2与建兰(Cymbidium ensifolium)CeCOL(90.77%)高度相似;OnCOL8与OnCOL9在进化关系上更为接近,分别与拟南芥(Arabidopsis thaliana)AtCOL9和AtCOL10关系最近,它们在B-box和CCT结构域都极为保守。表达分析结果表明OnCOL2、OnCOL8与OnCOL9分别在花、根和假鳞茎中表达量最高,在叶片中的表达呈周期性变化趋势,且在长、短日照条件下表达的峰值及时间均存在差异,在花芽分化时期的叶片中表达量均显著上调。研究表明,OnCOL2、OnCOL8与OnCOL9基因均受到生物钟和日长调节,在光周期途径中,可能通过上调它们的表达以促进文心兰花芽的形成。该研究结果为进一步研究基因功能及光周期调控文心兰开花机制奠定了基础。     

绿带闭目天蛾和构月天蛾在海南岛及周边大陆的遗传分化

海南岛是与周边大陆存在地理隔离的大陆性岛屿,探索该岛屿与邻近大陆上生物的遗传分化有助于理解地理隔离对生物遗传分化的影响。在海南岛及其周边大陆15个地点采集两种鳞翅目天蛾科(Lepidoptera: Sphingidae)昆虫(绿带闭目天蛾Callambulyx rubricosa 88头和构月天蛾Parum colligata 57头),选择线粒体基因COI、Cytb和核基因 EF-1α作为分子标记开展遗传多样性研究。基于COI-Cytb分析的结果表明,两物种的核苷酸多样性均较低(分别为0.00434和0.00818),且均在广东广西、越南和海南岛群体之间存在明显的谱系地理结构。两个物种在三个群体间均检测出低水平的基因流(N_m分别为0.09-1.22和0.18-0.57)和高水平的遗传分化(F_ST分别为0.29-0.84和0.47-0.73)。两个物种最高的遗传分化都发生在海南岛与大陆群体之间,且海南岛群体的单倍型数量少于周边大陆群体。Mantel检验结果表明遗传距离与地理距离之间存在显著正相关关系,中性检验结果表明两个物种均未经历显著的种群扩张。综上,研究认为绿带闭目天蛾(C. rubricosa)和构月天蛾(P. colligata)的海南岛群体与邻近大陆群体间的基因交流较少,遗传分化程度较高,推测地理隔离可能是其产生遗传分化的重要原因之一,这一结果将为海南岛生物多样性的保护提供宝贵的建议。     

变温处理对西红花花芽分化及其生理生化的影响

以西红花球茎为材料,设置20℃-25℃-20℃(T1)、20℃-30℃-25℃(T2)、25℃-35℃-25℃(T3)及25℃-30℃25℃(经验变温对照,CK1)和25℃25℃25℃(恒温对照,CK2)共5组三阶段(Ⅰ-Ⅱ-Ⅲ)变温处理,在各阶段采集球茎样品,用石蜡制片方法观察西红花花芽的分化情况,并测定各处理组球茎...     

Evolutionary relationships among aquatic anamorphs and teleomorphs: Lemonniera, Margaritispora, and Goniopila

The hypothesis that similar conidial morphologies in aquatic hyphomycetes are a result of convergent evolution was tested using molecular sequence data. Cladistic analyses were performed on partial sequences of 28S rDNA of seven species of Lemonniera, one species of Margaritispora and one species of Goniopila. Lemonniera has tetraradiate conidia with long arms, whereas Margaritispora and Goniopila have typically globose (isodiametric) conidia, with short conical protuberances in a stellate or quadrangular arrangement. Lemonniera and Margaritispora have phialidic conidiogenesis and both produce dark, minute sclerotia in culture whereas Goniopila has holoblastic conidiogenesis and does not produce sclerotia in culture. Goniopila produces a microconidial phialidic synanamorph in culture. All three genera have schizolytic conidial secession. Molecular analyses demonstrate that Lemonniera species are placed in two distinct clades: one within Leotiomycetes; the other within Pleosporales, Dothideomycetes. Margaritispora is placed with Lemonniera species within Leotiomycetes. Goniopila and Lemonniera pseudofloscula are placed within Dothideomycetes. No morphological character was entirely congruent with the molecular derived phylogeny. This suggests that for the group of species studied, conidial shape is not a reliable indicator of phylogeny but more likely the result of convergent evolution in response to the aquatic environment.     

Characterisation of IS153, an IS3-family Insertion Sequence Isolated from Lactobacillus sanfranciscensis and its use for Strain Differentiation

An insertion sequence has been identified in the genome of Lactobacillus sanfranciscensis DSM 20451^T as segment of 1351 nucleotides containing 37-bp imperfect terminal inverted repeats. The sequence of this element encodes two out of phase, overlapping open reading frames, orfA and orfB, from which three putative proteins are produced. OrfAB is a transframe protein produced by –1 translational frame shifting between orf A and orf B that is presumed to be the transposase. The large orfAB of this element encodes a 342 amino acid protein that displays similarities with transposases encoded by bacterial insertion sequences belonging to the IS3 family.In L. sanfranciscensis type strain DSM 20451^T multiple truncated IS elements were identified. Inverse PCR was used to analyze target sites of four of these elements, but except of their highly AT rich character not any sequence specificity was identified so far. Moreover, no flanking direct repeats were identified. Multiple copies of IS 153 were detected by hybridization in other strains of L. sanfranciscensis. Resulting hybridization patterns were shown to differentiate between organisms at strain level rather than a probe targeted against the 16S rDNA. With a PCR based approach IS 153 or highly similar sequences were detected in L. acidophilus, L. casei, L. malefermentans, L. plantarum, L. hilgardii, L. collinoides L. farciminis L. sakei and L. salivarius, L. reuteri as well as in Enterococcus faecium, Pediococcus acidilactici and P. pentosaceus.