Cloning and Characterization of New Repetitive Sequences in Field Bean (Vicia fabaL.)

Five new repetitive sequences have been isolated from theViciafabagenome, by cloning bands visible on agarose gel electrophoresisafter digestion of genomic DNA with various restriction enzymes.The sequences were 109 to 584 bp long, their abundance rangingfrom 5x10⁴to 5x10⁵copies per haploid genome. Southern blot andinsituhybridization revealed that four of five newly isolatedrepeats were dispersed in theV. fabagenome. One of the repeats(TIII15) showed tandem organization with several major hybridizationspots on mitotic chromosomesin situ.These sites were distributedin euchromatic as well as in heterochromatic chromosomal regions,and in several loci they were simultaneously localized withpreviously describedFokI repeated elements. The sequence ofTIII15 comprises four 26–27 bp subrepeats, but sharesno homology toFokI elements which have similar sequence organization.All newly described sequences were highly specific forV. faba,withlittle or no hybridization to DNA of otherViciaspecies, andno hybridization to DNA of other legumes tested.Copyright 1999Annals of Botany Company Vicia faba, field bean, repeated DNA sequences, FISH, PRINS, genome organization, copy number.     

Restriction Enzyme-Resistant High Molecular Weight Telomeric DNA Fragments in Tobacco

Restriction endonuclease-resistant high-molecular-weight (HMW)DNA fragments were isolated from nuclear DNA fragments in tobacco.The size of the fragments produced by EcoRI, HindIII, AfaI,and HaeIII ranged from 20 kb to over 166 kb. The kinetics ofdigestion by Bal31 nuclease showed that most of the HMW fragmentsare chromosome ends. The consensus sequence for tobacco telomererepeats was determined to be CCCTAAA by genomic sequencing usingthe HMW fragments and by sequencing after cloning. Besides thetelomere sequence, 9 tandem repeats of a 45-bp sequence wereidentified, in which a 35-bp unit sequence (AGTCAGCATTAGGGTTTTAAACCCTAAACTGAACT)formed a stem structure. The front of the stem is composed ofa palindrome of the telomere repeats. This highly conservedunit is surrounded by less conserved internal sequences thatare around 10–11 bp in size and contain a TTTT stretch.The internal sequences resemble the 10–11 bp consensusfor the scaffold attachment regions found in yeast and drosophila.The characteristic 45-bp sequence was abundant on the ends ofchromosomes. The shortest distance between the repeats containingtelomeric stem and the telomere was less than 20 kb. This architectureof the tobacco chromosome end region resembles the end regionof yeast chromosomes in which autonomous replication sequencesare present frequently.     

中国黄粉蝶亚科六属间基于COⅡ和EF-1α基因部分序列的系统发育关系（鳞翅目：粉蝶科）

为了探讨中国黄粉蝶亚科属间的系统发育关系,我们对其中6属9种的细胞色素氧化酶Ⅱ（COⅡ）的部分序列和延伸因子基因（EF-1α）部分序列进行了分析。分别采用最大简约法（maximum parsimony, MP）、最大似然法（maximum likelihood, ML）和贝叶斯推论法（bayesian inference, BI）构建黄粉蝶亚科分子系统树。结果表明：在测得的COⅡ基因的648 bp序列和EF-1α基因的504 bp序列中,有261个变异位点,151个简约信息位点,黄粉蝶亚科内各属COⅡ基因A+T含量（77.3%）均明显偏高。系统发育分析显示黄粉蝶属为亚科中较为原始的类群,分化较早,豆粉蝶属和迁粉蝶属亲缘关系较近,但钩粉蝶属与豆粉蝶属、迁粉蝶属之间的亲缘关系还不能确定。本研究结果和传统的基于形态学的黄粉蝶亚科的分类体系有所不同,最显著的分歧是本研究支持内群中分化最早的属应为黄粉蝶属,而不是豆粉蝶属和迁粉蝶属。     

感染稻水象甲的Wolbachia基因组中插入序列的鉴定与分析

共生细菌Wolbachia对宿主的生殖起多种调控作用。以往研究表明, Wolbachia基因组中广泛存在插入序列(insertion sequence, IS), 它们对宿主基因组的可塑性、 多样性和进化起重要作用。稻水象甲Lissorhoptrus oryzophilus Kuschel在东亚是一种外来水稻害虫, 在原产地北美营两性生殖, 而在所有入侵地均营孤雌生殖。本研究采用PCR法从河北唐海孤雌生殖型稻水象甲体内克隆获得了Wolbachia的2条IS序列, 即ISWosp4和ISWosp6; 从美国德克萨斯州两性生殖型稻水象甲成虫体内克隆获得了Wolbachia的2条IS序列, 即ISWosp3和ISWosp5。碱基序列比对显示: ISWosp3和ISWosp4属于IS3家族IS3组成员, ISWosp5为IS4家族IS231组成员, ISWosp6为IS5家族IS1031组成员。对这些IS的ORF结构、 所编码氨基酸序列的结构等进行了分析, 推测ISWosp5具有潜在转座活性。所得结果增进了我们对Wolbachia IS3, IS4和IS5家族插入序列的认识, 同时为今后从IS的角度探讨Wolbachia与稻水象甲生殖的关系奠定了基础。     

An integrated genetic linkage map of avocado

 An avocado genomic library was screened with various microsatellite repeats. (A/T)_n and (TC/AG)_n sequences were found to be the most frequent repeats. One hundred and seventy-two positive clones were sequenced successfully of which 113 were found to contain simple sequence repeats (SSR). Polymerase chain reaction primers were designed to the regions flanking the SSR in 62 clones. A GenBank search of avocado DNA sequences revealed 1 sequence containing a (CT)₁₀ repeat. A total of 92 avocado-specific SSR markers were screened for polymorphism using 50 offspring of a cross between the avocado cultivars ‘Pinkerton’ and ‘Ettinger’. Both are standard avocado cultivars which are normally outcrossed and highly heterozygous. Fifty polymorphic SSR loci, 17 random amplified polymorphic DNA (RAPD) and 23 minisatellite DNA Fingerprint (DFP) bands were used to construct the avocado genetic map. The resulting data were analyzed with various mapping programs in order to assess which program best accommodated data from progeny of heterozygous parents. The analyses resulted in 12 linkage groups with 34 markers (25 SSRs, 3 RAPDs and 6 DFP bands) covering 352.6 cM. This initial map can serve as a basis for developing a detailed genomic map and for detection of linkage between markers and quantitative trait loci. Received: 2 April 1996 / Accepted: 28 February 1997     

家蝇幼虫消减文库的构建及差异表达基因的鉴定

为了鉴定家蝇Musca domestica免疫相关基因,应用抑制性消减杂交技术,构建刺激家蝇幼虫差异表达cDNA消减文库。以大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus诱导12 h的家蝇幼虫与未诱导的家蝇幼虫为消减杂交对象,获得了差异表达基因的cDNA片段,将其与T/A载体连接并转化大肠杆菌DH5α,构建了刺激家蝇幼虫cDNA消减文库。PCR检测发现,文库的阳性克隆中插入的cDNA片段大小在200~1 000 bp之间,随机挑选了161个含大小不等差异片段的克隆进行测序和同源性分析,鉴定了36种蛋白的基因片段,包括抗菌肽、酶、核糖体蛋白、其他功能蛋白以及功能不明的蛋白。用半定量RT-PCR分析了其中6种蛋白基因的表达,结果显示：防御素和攻击素基因在细菌刺激后24 h内明显上调表达,而溶菌酶、酚氧化酶原活化因子、糜蛋白酶和蛋白质合成起始因子基因在细菌刺激后0-4 h内表达受抑制,12 h后上调表达。该研究结果为家蝇免疫相关基因的克隆和家蝇免疫防御机制的探讨奠定了良好的基础。     

水稻褐飞虱内生共生细菌Arsenophonus的鉴定和系统分析

利用16S rDNA通用引物扩增了水稻褐飞虱Nilaparvata lugens（Stål）体内共生细菌的序列,经克隆、测序和NCBI数据库比对,发现褐飞虱体内存在杀雄菌属Arsenophonus类共生细菌,系统发育上与粉虱科和木虱科体内的Arsenophonus属亲源关系较近。在褐飞虱体内该共生细菌具有两种长度不同的16S rDNA序列,分别为1 504 bp和547 bp,其中后者为前者中间缺失了957 bp,其余序列相同。通过重新设计两对引物进行扩增,进一步确认不同褐飞虱地理种群及寄主种群均存在两种片段。Arsenophonus特异的 23S rDNA引物的扩增结果表明,Arsenophonus存在于所有检测的褐飞虱种群中,但不存在于水稻寄主中。荧光定量PCR检测发现3个褐飞虱室内寄主种群Arsenophonus属共生细菌含量不同,其中TN1种群明显高于Mudgo种群和ASD7种群。此为水稻褐飞虱体内存在Arsenophonus属共生细菌的首次报道。     

中国部分地区柑桔木虱体内感染Wolbachia的检测及其系统发育分析

Wolbachia是一种广泛分布于节肢动物体内的共生细菌, 该共生菌经寄主卵的细胞质传播并参与多种寄主生殖活动调控, 对节肢动物的物种进化有着重要意义并可能应用于害虫的生物防治。本研究以柑桔黄龙病（Huanglongbing, HLB）的主要传播媒介——柑桔木虱Diaphorina citri为研究对象, 通过Wolbachia的16S rDNA、 细胞分裂基因（ftsZ）、 表面蛋白基因（wsp）的特异引物对来自于中国广西北海、 广西柳州、 广东深圳、 广东阳春、 福建福州5个地区的柑桔木虱进行PCR扩增, 并对扩增产物进行克隆测序, 与GenBank中相关序列进行比对分析, 建立了柑桔木虱共生Wolbachia的系统发育树。结果显示上述5个地区的柑桔木虱均存在Wolbachia感染, 通过Wolbachia的16S rDNA, ftsZ基因和wsp基因DNA序列的系统发育分析表明, 5个地区的柑桔木虱体内的Wolbachia均属于B组; 基于wsp基因的系统发育分析进一步表明5个地区的亚洲柑桔木虱体内的Wolbachia属于B组的Con亚组, 且不同地区柑桔木虱体内的Wolbachia的16S rDNA, ftsZ基因和wsp基因序列差异不明显。研究结果为认识柑桔黄龙病传播媒介昆虫的进化及其综合防治提供了重要的参考依据。     

B型烟粉虱和温室白粉虱热激蛋白90基因（hsp90）的全长cDNA克隆与系统发育分析

B型烟粉虱Bemisia tabaci (Gennadius) biotype B和温室白粉虱Trialeurodes vaporariorum均为全球普遍发生的重要害虫。本研究以其他昆虫热激蛋白90基因（hsp90）保守区域设计兼并引物扩增两种粉虱hsp90中间片段, 然后利用RACE技术获得全长cDNA。温室白粉虱hsp90全长cDNA的开放性阅读框2 166 bp, 编码722个氨基酸; 烟粉虱hsp90全长cDNA的开放性阅读框2 160 bp, 编码720个氨基酸。两种粉虱HSP90的完整氨基酸序列相似性高达92.94％, 并均具有定义HSP90家族签名序列的5个氨基酸保守区域和末尾基序“MEEVD”。通过real-time PCR技术, 探测到两个基因在mRNA水平上皆能高温诱导表达。采用昆虫纲所有完整HSP90氨基酸序列进行Kimura双参数遗传距离分析并构建NJ进化树, 结果显示hsp90在昆虫纲低级阶元水平和高级阶元水平系统进化上能得到一个较理想结果。本研究结果为B型烟粉虱和温室白粉虱抗逆适应性研究提供基础, 并进一步验证保守的功能基因hsp90可以作为研究生物系统发育的手段之一     

华北大黑鳃金龟两种围食膜蛋白cDNA的分子克隆与序列分析

本文以华北大黑鳃金龟Holotrichia oblita中肠为材料,依据Stratagene公司文库构建试剂盒方法,构建其中肠cDNA表达文库,该文库滴度为1.9×106 pfu/mL,重组率为99.97%。依据现代免疫学原理,利用棉铃虫Helicoverpa armigera围食膜蛋白多克隆抗体筛选文库,得到两个编码华北大黑鳃金龟围食膜蛋白的cDNA克隆Ho-Peritrophin1与Ho-Peritrophin2,其cDNA长分别为2 385 bp和1 633 bp,在PolyA末端上游各有3个多聚腺苷酸信号序列AATAAA,最长开放阅读框(ORF)分别编码729个和477个氨基酸,与粉纹夜蛾Trichoplusia ni CBP2(chitin binding protein 2)的相似性最高,分别为21.9%和19.1%。结构域分析表明,Ho-Peritrophin1与Ho-Peritrophin2分别具有9个和6个几丁质结合功能域,只含有较少的O-糖基化位点,不含有类粘蛋白结构域。胰蛋白酶和胰凝乳蛋白酶对两种蛋白的作用位点主要位于几丁质结合功能域(chitin binding domain, CBD)内部,而因受几丁质结合功能域保护,这两种蛋白能够抵抗这些蛋白酶的降解。与正常CBD比较,这两种蛋白C端的CBD只含有4个Cys,只在第1与第3、第4与第5个Cys之间形成两对二硫键,缺少由第2与第6个Cys形成的二硫键。推测其N端还应包括信号肽序列和几丁质结合功能域的未知序列。     

基于线粒体COI基因序列的文蛤属(软体动物门:帘蛤科)系统发育关系(英文)

测定了4种文蛤属贝类的15个个体的COI基因序列,并从GenBank下载了短文蛤 (M. petechialis)的相应序列。比对后的序列长度为574 bp,包括93个简约信息位点,A、T、C和G的平均含量分别为21.15%、44.71%、14.05%和20.09%。通过对序列的分析,共定义了12个单倍型：文蛤 (M. meretrix) 4个,斧文蛤 (M. lamarckii) 2个,丽文蛤 (M. lusoria) 3个,琴文蛤 (M. lyrata) 1个,短文蛤2个。以青蛤(Cylina sinensis)为外群,用MP法和贝叶斯法构建系统树。结果显示,短文蛤、丽文蛤和文蛤为亲缘关系较近的物种, 支持短文蛤和丽文蛤为文蛤的同物异名的观点。     

Repetitive DNA, Genome and Species Relationships in Avena and Arrhenatherum(Poaceae)

Repetitive sequences have been widely used for examining genomeand species relationships by in situ and Southern hybridization.In the present study, double-stranded DNA sequences, from denaturedDNA reannealed to Cot = 1, from Avena strigosa(2 n = 2x = 14;A genome; referred to as CotA) and Avena sativa(2n = 6 x =42; ACD genome; referred to as CotACD) were isolated with ahydroxyapatite column, and were used for in situ hybridizationon hexaploid A. sativa chromosomes. Probe CotACD labelled allchromosomes evenly throughout their length at the same intensity.Probe CotA labelled the 28 A and D genome chromosomes stronglyand the 14 C genome chromosomes weakly. Three cloned repetitivesequences, pAvKB9 (126 bp), pAvKB26 (223 bp) and pAvKB32 (721bp) were characterized in the A, B, C and D Avena genomes andthe genus Arrhenatherum using molecular and cytological methods.Clones pAvKB9 and pAvKB26 were absent from the Avena C genome,while both could identify the presence of the D genome by Southernhybridization. In situ hybridization to diploid and tetraploidAvena species revealed that the probes showed a dispersed genomicorganization and that they are present on both arms of all chromosomes.These sequences were excluded from areas where tandem repeats,such as rRNA genes and telomeres, are present. These resultsindicate the close relationship between A and D genomes andthe presence of common DNA sequences between A and C Avena genomes.All three clones hybridized to Southern blots containingArrhenatherumdigested genomic DNA, indicating Arrhenatherum’s closeaffinity to A, B and D Avena genomes. Copyright 2000 Annalsof Botany Company Cereals, DNA, hydroxyapatite, in situ hybridization, oats, reassociation kinetics, repetitive DNA     

Effects of temperature on the cytokinin requirement of tobacco calluses

A cytokinin-nonrequiring strain (T22) was isolated from a cytokinin-requiringcallus strain T2 of tobacco (Nicotiana tabacum var. Bright Yellow). Strain T22 grew rapidly on the medium without added kinetinat 26?C. But its growth was completely suppressed at 16?C. Thisgrowth suppression at 16?C was partially recoverable by supplyingkinetin. Benzyladenine, geranylaminopurine and 2-methyl-8-benzylamino-s-triazolo[l,5-a]pyrazinewere also effective in removing growth suppression at 16?C.Adenine, which was unable to remove growth suppression of T22at 16?C, promoted the growth of T2 at 26?C, but not at 16?C.Physiological differences between cytokinin-requiring and -nonrequiringcalluses are discussed. ¹Part II in the series "Studies, on Plant Tissue Cultures";for Part I, See Plant & Cell Physiol. 9: 103–114 (1968). (Received May 29, 1970; )     

AIMIE: a web-based environment for detection and interpretation of significant sequence motifs in prokaryotic genomes

Motivation: Genomes contain biologically significant informationthat extends beyond that encoded in genes. Some of this informationrelates to various short dispersed repeats distributed throughoutthe genome. The goal of this work was to combine tools for detectionof statistically significant dispersed repeats in DNA sequenceswith tools to aid development of hypotheses regarding theirpossible physiological functions in an easy-to-use web-basedenvironment. Results: Ab Initio Motif Identification Environment (AIMIE)was designed to facilitate investigations of dispersed sequencemotifs in prokaryotic genomes. We used AIMIE to analyze theEscherichia coli and Haemophilus influenzae genomes in orderto demonstrate the utility of the new environment. AIMIE detectedrepeated extragenic palindrome (REP) elements, CRISPR repeats,uptake signal sequences, intergenic dyad sequences and severalother over-represented sequence motifs. Distributional patternsof these motifs were analyzed using the tools included in AIMIE. Availability: AIMIE and the related software can be accessedat our web site http://www.cmbl.uga.edu/software.html. Contact: mrazek{at}uga.edu Associate Editor: Alex Bateman     

卷蛾分索赤眼蜂和拟澳洲赤眼蜂对小菜蛾利他素各成分的嗅觉反应

卷蛾分索赤眼蜂Trichogrammatoidea bactrae Nagaraja和拟澳洲赤眼蜂Trichogramma confusum Viggiani是小菜蛾Plutella xylostella (L.)的优势寄生蜂。本研究利用“Y”型管测定了2种赤眼蜂对小菜蛾P. xylostella利他素-卵表和腹部鳞片13种饱和烷烃的嗅觉反应。结果显示: 卷蛾分索赤眼蜂交配雌蜂进入2, 6, 10, 14-四甲基十五烷、正十五烷、正十七烷处理区的百分数分别为80.65%, 68.75%和66.67%, 表明这3种烷烃对卷蛾分索赤眼蜂交配雌蜂有显著的吸引作用, 其他10种饱和烷烃对卷蛾分索赤眼蜂雌蜂无明显作用。拟澳洲赤眼蜂进入2, 6, 10, 14-四甲基十五烷、正三十五烷和正十五烷处理区的数目分别占84.38%, 70%和62.16%, 表明其对拟澳洲赤眼蜂交配雌蜂起着显著的吸引作用, 而另外10种烷烃对拟澳洲赤眼蜂雌蜂无作用。卷蛾分索赤眼蜂和拟澳洲赤眼蜂未交配雄蜂进入13种烷烃处理区和对照区的时间均无显著差异, 表明利他素各组分对2种赤眼蜂未交配雄蜂均无吸引作用。     

Cloning and Mapping of Telomere-Associated Sequences from Rice

We have isolated three telomere-associated sequences from riceusing cassette-ligation-mediated polymerase chain reaction (PCR).Each of the obtained clones hybridized to the terminal of oneor several rice chromosome arms. The telomeres recognized bythe clones displayed a high level of polymorphism between tworice varieties, Nipponbare (a japonica variety) and Kasalath(an indica variety). Variability in the chromosome termini wasalso detected among individual F₂ progeny plants, which werederived from a cross between the two rice varieties. One clonecontaining telomere-associated sequences was located to oneend of chromosome 5, and another clone to one end of chromosome11. For another clone, non-allelic segregation of polymorphichybridization bands was observed between japonica and indicarice; this clone was mapped to one end of chromosome 12 in japonicaand to one end of chromosome 11 in indica rice. This indicatesan exchange of termini between nonhomologous chromosomes.     

昆明山海棠的杀虫活性及有效成分

【目的】从天然产物尤其是植物中寻找杀虫活性物质是杀虫剂创制的重要途径。【方法】采用生物活性追踪、分离等方法,研究了昆明山海棠Tripterygium hypoglaucum (Levl.) Hutch对6种鳞翅目昆虫的杀虫活性及有效成分。【结果】昆明山海棠根皮石油醚提取物、甲醇提取物和乙酸乙酯提取物对粘虫3龄幼虫均具有拒食活性,24 h的拒食毒力AFC₅₀值分别为1 165.7 μg/mL、104.3 μg/mL和 47.3 μg/mL。昆明山海棠根皮甲醇提取物对粘虫4龄幼虫具有触杀活性,其LD₅₀值为100.4 μg/头。从昆明山海棠根皮甲醇提取物中分离出雷公藤春碱、雷公藤吉碱、雷公藤定碱和雷公藤榕碱 4个杀虫活性化合物,雷公藤春碱和雷公藤吉碱对粘虫具有胃毒麻醉活性,对粘虫3龄幼虫8 h的麻醉中量（ND₅₀）分别是18.1 μg/头和7.4 μg/头。雷公藤定碱和雷公藤榕碱对粘虫具有触杀麻醉活性,对粘虫4龄幼虫24 h的ND₅₀分别是0.33 μg/头和0.06 μg/头;对3龄小地老虎具有胃毒麻醉活性,24 h的麻醉中量ND₅₀分别是5.62 μg/头和1.24 μg/头。【结论】昆明山海棠对所测试的6种鳞翅目昆虫均有一定的杀虫活性,其主要杀虫活性成分为雷公藤春碱、雷公藤吉碱、雷公藤定碱和雷公藤榕碱4个化合物。     

Localization of Purothionins and Genome Expression in Wheat Plants

The leaves, steins and ears of tetraploidal wheat, Triticumdurum (genomes AABB) harvested two weeks after heading wereanalyzed for purothionins. There were considerable amounts oftwo iso-purothionins (Pu-1 and Pu-2) in the young kernels ofthe ears, but not in the leaves and stems. The amino acid compositionsof Pu-1 and Pu-2 were in good agreement with those of Pu I andPu II from hexaploidal wheat. As diploidal wheat, T. monococcum(genome AA) is known to produce only Pu I [Fernandez de Caleyaet al. (1976) Genetics 83: 687], we conclude that the threeiso-purothionins, Pu I, Pu II and Pu III in hexaploidal wheat,T. aestivum (genomes AABBDD), are encoded by the genes of genomesA, B and D, respectively. (Received June 28, 1982; Accepted September 24, 1982)     

Sixty New STSs (Sequence-Tagged Sites) of Human Chromosome 21

From human chromosome 21-specific libraries, 22 SfiI linkingclones and 38 P1 clones were isolated and regionally mappedon the chromosome. The terminal sequences of these clones weredetermined and pairs of PCR primers were generated which couldspecifically amplify the sequenced regions. These sequence-taggedsites (STSs) should be useful for constructing a high resolutionmap of human chromosome 21.