中华蜜蜂的单核苷酸多态性和插入缺失突变位点鉴定

【目的】本研究拟利用已获得的中华蜜蜂Apis cerana cerana幼虫肠道的转录组数据对单核苷酸多态性（Single nucleotide polymorphism,SNP）和插入缺失（Insertion-Deletion,InDel）突变位点进行挖掘和分析,旨在丰富中华蜜蜂的SNP和InDel信息,并为新型分子标记的开发提供基础。【方法】根据有效读段与东方蜜蜂Apis cerana参考基因组的比对情况,采用GATK软件识别单碱基错配和碱基的插入缺失情况,再利用ANNOVAR软件对SNP位点和InDel位点进行分析。通过相关生物信息学软件将SNP和InDel位点所在基因分别比对GO和KEGG数据库,以获得相应的功能和通路注释。【结果】共鉴定到中华蜜蜂的58 919个SNP位点,包括24 548个纯合位点和34 371个杂合位点;发生转换和颠换的SNP位点分别有49102和9817个;数量最多和最少的突变类型分别是C/T和T/G;分布在外显子区的SNP位点数量最多,达到22 649个;此外,发生同义突变的SNP位点数量最多,其次是非同义突变;SNP位点所在基因可注释到46个GO条目和121条KEGG通路。共鉴定到6 551个InDel位点,包括3 270个插入突变和3 281个缺失突变;分布在内含子区InDel位点最多,共计2 793个;发生移码插入的InDel位点最多;进一步分析结果显示InDel位点所在基因可注释到27个GO条目和28条KEGG通路。【结论】本研究鉴定到中华蜜蜂的大量SNP位点和InDel位点,解析了SNP和InDel位点的突变类型、基因组功能元件分布和密码子突变类型,并揭示SNP和InDel位点对中华蜜蜂的重要生物学过程具有潜在影响。     

东方蜜蜂微孢子虫的SNP与InDel位点鉴定及分析

【目的】东方蜜蜂微孢子虫Nosema ceranae是一种专性侵染成年蜜蜂中肠上皮细胞的单细胞真菌病原,广泛感染世界各地的蜂群。本研究拟利用已获得的东方蜜蜂微孢子虫纯净孢子的高质量转录组数据进行单核苷酸多态性（Single nucleotide polymorphism,SNP）和插入缺失（Insertion-Deletion,InDel）位点的鉴定和分析,旨在丰富东方蜜蜂微孢子虫的SNP和InDel信息,并为新型分子标记的开发提供基础。【方法】使用GATK软件识别东方蜜蜂微孢子虫的SNP和InDel位点。采用SnpEff软件预测变异位点发生的基因组区域及变异产生的影响。通过相关生物信息学软件分别将SNP和InDel位点所在基因分别比对GO和KEGG数据库,获得相应的功能和通路注释。【结果】共鉴定到28195个SNP位点,其中发生转换和颠换的SNP位点分别有21403和6792个;上述SNP位点的突变类型有12种,其中最丰富的突变类型为C/T;分布在CDS区的SNP位点最多,其次是基因间区、上游区、下游区和内含子区;最丰富的密码子突变类型是同义突变;SNP位点所在基因可注释到代谢进程、细胞组分和催化活性等43个GO条目以及代谢途径、核糖体和等次生代谢产物的生物合成等85条KEGG通路。共鉴定到2831个InDel位点,其中分布在基因间区InDel位点最多,分布在CDS区的InDel位点最少;最丰富的密码子突变类型是移码突变;InDel位点所在基因可注释到细胞进程、细胞和结合等38个GO条目以及代谢途径、次生代谢产物的生物合成及核糖体等73条KEGG通路。【结论】东方蜜蜂微孢子虫中存在大量的SNP和InDel位点,SNP位点的突变类型主要为转换,与其他物种类似;SNP与InDel位点的基因组功能元件分布规律和突变类型具有明显差异;SNP和InDel位点所在基因与东方蜜蜂微孢子虫适应宿主细胞内环境及病原增殖过程具有潜在关系。     

基于BSA-seq法的油菜野芥胞质雄性不育恢复基因的分析

细胞质雄性不育是利用杂种优势创制油菜杂交种的重要途径之一。而恢复系的选育及恢复基因的研究有利于提高细胞质雄性不育系的应用价值。该研究以甘蓝型油菜细胞质雄性不育系1193A和恢复系15-R1为亲本,以F_2群体构建了2个极端性状混池,采用BSA-seq技术方法,对恢复基因进行定位分析。结果表明:(1)不育系1193A与恢复系15-R1之间共获得非同义突变的SNPs共33 883个,混池间共有7 996个非同义编码突变。(2)InDel检测与注释结果表明,亲本间引起移码突变的有2 918个,混池间引起移码突变的有840个。(3)综合SNP和InDel的关联分析结果,将恢复基因定位于C09染色体上的0～880000区域,总长度为880 kb。(4)对候选区域的SNP和InDel注释发现,亲本间存在非同义突变的SNP共40个,混池间存在非同义突变的SNP共65个,亲本间存在移码突变的InDel共7个,混池间存在移码突变的InDel共11个;对候选区间内的编码基因进行注释,结果共注释到162个基因,其中存在非同义突变基因11个,移码突变基因5个,这些基因可能与育性恢复有关。     

p38 MAPK基因在西方蜜蜂越冬期表达模式的研究

[目的]p38 MAPK基因在昆虫的低温响应机制中发挥着重要作用,本研究旨在探究p38 MAPK基因在西方蜜蜂Apis mellifera越冬期内表达规律.[方法]本研究对西方蜜蜂p38 MAPK蛋白序列进行生物信息学分析,并利用荧光定量PCR技术检测该基因在意大利蜜蜂Apis mellifera ligustica、欧洲黑蜂Apis mellifera mellifera、高加索蜂Apis mellifera caucasica和卡尼鄂拉蜂Apis mellifera carnica于不同越冬时期和不同越冬方式下体内的表达量.[结果]西方蜜蜂38 MAPK包含1083 bp的开放阅读框区域,编码360个氨基酸,包含TGY双磷酸化三肽模体序列和磷酸化激活环序列.p38 MAPK在蜜蜂所有组织中均有表达,分别在胸部和腹部处于最高和最低表达丰度.38 MAPK mRNA在不同蜂种不同越冬时期的表达量存在显著差异,4个蜂种于室外越冬时p38 MAPK的表达量均显著高于室内越冬(P＜0.05);随着蜜蜂越冬持续时间的延长,意大利蜜蜂和欧洲黑蜂不同越冬方式下体内p38 MAPK的表达均呈现先上升后下降的表达趋势,高加索蜂在室外与室内越冬时体内该基因的表达量均表现出持续上升的表达趋势,该基因在卡尼鄂拉蜂中的表达趋势与其他3个蜂种呈现一定差异,卡尼鄂拉蜂p38 MAPK在室内越冬方式下表达量保持恒定,但在室外越冬方式下表现出先下降后上升的表达趋势;比较室外越冬情况下4个蜂种于不同越冬时期体内38 MAPK表达量后发现,除在次年的1月外,其他越冬时期4个蜂种体内p38 MAPK的表达量均存在显著差异(P＜0.05).[结论]p38 MAPK基因在西方蜜蜂越冬期内发挥着重要的生理功能,p38 MAPK信号通路可作为蜜蜂抗寒机制研究的候选信号通路.     

引入西方蜜蜂对中蜂的危害及生态影响

作者阐述自1896年中国引进西方蜜蜂Apis melliferaL.的优良品种如意大利蜂Apis mellifera ligusticaSpinola和喀尼阿兰蜂Apis mellifera Carnica Pollmann以来,使当地的东方蜜蜂Apis cerana F.受到严重危害,其分布区域缩小75%以上,种群数量减少80%以上。使山林植物授粉总量减少,导致植物多样性减少。文中提出: 建立自然保护区保存本地蜜蜂遗传特性,和采用基因转移等技术,培育具有西方蜜蜂优良生产性能的中蜂新品种,逐步恢复中蜂的种群数量。     

人类基因组致病SNP与良性SNP分布模式探究

基因组变异与生物学功能,乃至人体健康紧密相关。不同种类的基因组变异会对人类健康产生不同的影响,可能出现致病、良性或未知的临床症状。深入研究临床上表现为致病性或良性的基因组变异,能够有效加深我们对基因组变异与疾病发生发展的认识,对评估基因组变异所带来的影响具有一定的意义。本研究选择Clinvar数据库中与疾病相关的SNP,探究临床表现为致病与良性的SNP位点分别在基因组上的分布模式以及对蛋白序列的影响。本研究发现,致病SNP与良性SNP在外显子与内含子上的分布以及蛋白结构域内外的分布上具有非常显著差异,致病SNP在外显子和蛋白结构域内的分布密度更高。从蛋白编码区的SNP突变类型来看,致病SNP位点中的同义突变与非同义突变所占比例分别是0.64%和99.36%,而良性SNP位点中两类突变的比例相差较小,检验结果显示致病SNP位点中非同义突变更多。此外,本研究通过分析致病SNP与良性SNP中的同义突变与非同义突变在蛋白质结构域内外的分布情况,发现两类SNP变异中同义突变的分布没有显著差异,但非同义突变的分布具有显著差异,其中致病SNP变异分布在蛋白质结构域内外的比例是53.22%和46.78%,良性SNP变异分布比例为31.96%和68.04%。随后,我们对非同义突变所在基因进行了功能注释和富集分析,发现两类变异所在基因的功能范围几乎一致。     

蜜蜂表皮蛋白apidermin (apd)基因家族3个新成员的特性鉴定及昆虫APD家族序列特征的分析

Apidermin (APD)蛋白家族是一个新的昆虫结构性表皮蛋白家族。本研究结合生物信息学和RT-PCR扩增, 对意大利蜜蜂Apis mellifera ligustica（简称“意蜂”）的apd-1-like, apd-3-like和中华蜜蜂Apis cerena cerena（简称“中蜂”）的apd-2 等3个新的apd基因的结构特征和表达进行了分析, 并分析了昆虫APD蛋白家族的序列特征。结果显示, 在西方蜜蜂Apis mellifera（简称“西蜂”）中, apd基因家族的6个成员串联排列在基因组序列第4号连锁群上, 它们在A. m. ligustica雄蜂头部中的转录水平差异明显, 且其启动子序列所含顺式元件也不同。中蜂apd-2和意蜂apd-1-like都含有3个外显子和2个内含子, 而意蜂apd-3-like则由4个外显子和3个内含子组成。蛋白序列分析结果显示, 目前已知的10条APD蛋白序列N末端均具有相似的信号肽序列, 其成熟蛋白分子量为6.0~37.0 kD, pI为6.2~10.8。其中西蜂的APD1-3、APD-like和东方蜜蜂Apis cerena的APD-2等5条较短的多肽中疏水氨基酸残基达52%~67%, 且Ala含量最为丰富（占25%~34%）; 而丽蝇蛹集金小蜂Nasonia vitripennis的APD 1-3和西蜂APD-1-like, APD-3-like等另外5条APD多肽富含Gly（21%~30%）, 其序列中疏水氨基酸残基含量为35%~41%。多肽序列多重比对和系统进化分析结果显示, APD家族可划分为2个亚家族。亚家族Ⅰ含有西蜂APD 1-3和东方蜜蜂APD-2等4条较短的多肽序列, 其N末端为一个长33 aa的保守基序; 亚家族Ⅱ由另外6条相对较长的多肽序列组成, 其N末端保守基序长50 aa, C末端保守基序长16 aa。本文所描述的APD蛋白家族序列特征有助于以后从其他昆虫中鉴定新的apd基因。     

基于微卫星标记的饶河黑蜂亲缘关系分析

为了摸清我国饶河黑蜂(亦称东北黑蜂)群体与俄罗斯蜜蜂(亦称USDA-ARS俄罗斯蜜蜂)、C形态学分支的卡尼鄂拉蜂Apis mellifera carnica、O形态学分支的高加索蜂Apis mellifera caucasica和安拉托尼亚蜂Apis mellifera anatoliaca之间的亲缘关系,本试验运用16个微卫星DNA标记,以来自饶河县7个区域(东北黑蜂国家级自然保护区所在地)的东北黑蜂为研究对象,其它5个蜜蜂群体为对照,分析了东北黑蜂与其他西方蜜蜂的亲缘关系、群体遗传结构和种质特性。结果表明东北黑蜂群体与俄罗斯蜜蜂亲缘关系最近(遗传距离(dμ)2=0.31～0.55),在遗传进化关系上处于同一进化分支,而与高加索蜂、安拉托尼亚蜂和卡尼鄂拉蜂的亲缘关系相对较远(遗传距离(dμ)2=0.52～0.86)。该实验结果进一步证明东北黑蜂起源于俄罗斯蜜蜂,其对东北黑蜂的选育、推广和保护具有重要的现实意义。     

中华蜜蜂mrjp1 cDNA的克隆及其序列分析

构建中华蜜蜂(Apis cerana cerana)8日龄工蜂头部cDNA文库,利用中蜂基因组的mrjp3部分基因片段作为杂交探针,采用DIG标记筛选cDNA文库,获得mrjps阳性克隆120个;对阳性克隆进行PCR扩增和测序,通过NCBI的BLAST序列比对,获得12个与印度蜂(Apis cerana india)、西方蜜蜂(Apis mellifera L.)mrjp1基因同源的中蜂mtjp1 cDNA片段,并进一步对中华蜜蜂mrjp1的cDNA全序列进行测定和分析。序列比对分析表明,东方蜜蜂(Apis cerana)与西方蜜蜂mrjp1的cDNA序列相似性为93．78％,中华蜜蜂与印度蜂的相似性高达99．36％,这一结果从分子水平证实中华蜜蜂与印度蜂有较近的共同祖先,而东方蜜蜂与西方蜜蜂的亲缘关系较远。     

中国意大利蜜蜂微卫星遗传多态性

为了分析我国不同生态条件下意大利蜜蜂Apis mellifera ligustica（简称意蜂）的基因特征,对品种间基因交流或基因渗入现象进行监测,完成对我国主要地区意蜂多样性水平的评价。本研究利用10对微卫星（SSR）引物分析了来自我国黑龙江等14个省市自治区31个采集点的意蜂样品的遗传多态性,并与来自德国的喀尼阿兰蜂A. m. carnica（简称喀蜂）和来自美国的意蜂A. m. ligustica进行比对。通过NTSYS-pc2.1和popGene32软件对SSR数据进行多态性分析。结果表明：10对SSR引物中8对表现出较高的遗传稳定性,可作为意蜂遗传多态性标记。遗传一致度在0.61以上时,浙江、四川、甘肃和云南地区的样品出现较严重分化,其他地区的意蜂样品则保持了美国原种意蜂的典型基因特征。东北和新疆地区的样品与德国喀蜂血统亲缘关系较近（遗传距离在0.18149以内）,分析主要原因可能是该地区的意蜂样品杂有黑蜂A. m. mellifera血统。据此提出应在保护各地意蜂原种特性及种群多态性的基础上,科学有效地进行高产品种的繁育。     

Copper (II) and cobalt (II) complexes of chiral tetrathiafulvalene-oxazoline (TTF-OX) and tetrathiafulvalene-thiomethyl-oxazoline (TTF-SMe-OX) derivatives

Synthesis and single crystal X-ray structures of the first paramagnetic transition metal complexes containing chiral ethylenedithio-tetrathiafulvalene-oxazoline (EDT-TTF-OX) 1a-c and ethylenedithio-tetrathiafulvalene-thiomethyloxazoline 2 (EDT-TTF-(SMe)OX) ligands based on copper (II) and cobalt (II) are described. The racemic [EDT-TTF-OX][Cu(hfac)₂] complex 3a crystallizes in the triclinic centrosymmetric space group , whereas the enantiopure counterparts 3b-c crystallize in the triclinic non-centrosymmetric space group P1. Cu(II) adopts a distorted square pyramidal coordination geometry, a much weaker Cu?S_TTF interaction also being identified. The same coordination pattern around Cu(II) is observed in the complex [(rac)-EDT-TTF-(SMe)OX][Cu(hfac)₂] (4) in spite of the bidentate nature of the redox active ligand. DFT theoretical calculations afforded two equilibrium configurations for a corresponding model complex, in which the metal centre establishes secondary coordination either with one S_TTF or with the SMe group. The same ligand coordinates the cobalt (II) to afford the octahedral complex [(rac)-EDT-TTF-(SMe)OX][Co(hfac)₂] (5). In all these novel complexes, the paramagnetic centres are structurally and magnetically isolated. Cyclic voltammetry measurements show the stability of the radical cation species.     

Binding of tetrakis(pyrazoliumyl)porphyrin and its copper(II) and zinc(II) complexes to poly(dG-dC)2 and poly(dA-dT)2

Interactions of cationic porphyrins bearing five-membered rings at the meso position, meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (MPzP; M is H₂, Cu^II or Zn^II), with synthetic polynucleotides poly(dG-dC)₂ and poly(dA-dT)₂ have been characterized by viscometric, visible absorption, circular dichroisim and magnetic circular dichroism spectroscopic and melting temperature measurements. Both H₂PzP and CuPzP are intercalated into poly(dG-dC)₂ and are outside-bound to the major groove of poly(dA-dT)₂, while ZnPzP is outside-bound to the minor groove of poly(dA-dT)₂ and surprisingly is intercalated into poly(dG-dC)₂. The binding constants of the porphyrin and poly(dG-dC)₂ and poly(dA-dT)₂ are on the order of 10⁶ M⁻¹ and are comparable to those of other cationic porphyrins so far reported. The process of the binding of the porphyrin to poly(dG-dC)₂ and poly(dA-dT)₂ is exothermic and enthalpically driven for H₂PzP, whereas it is endothermic and entropically driven for CuPzP and ZnPzP. These results have revealed that the kind of the central metal ion of metalloporphyrins influences the characteristics of the binding of the porphyrins to DNA.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Synthesis and properties of trinuclear polypyridyl complexes Ru(II)-Co(II)-Ru(II) and Ru(II)-Co(III)-Ru(II): Their photoinduced interconversion

The trinuclear [{Ru^II(bpy)₂(bpy-terpy)}₂Co^II]⁶⁺ complex (1⁶⁺) in which a Co(II)-bis-terpyridine-like centre is covalently linked to two Ru(II)-tris-bipyridine-like moieties by a bridging bipyridine-terpyridine ligand has been synthesised and characterised. Its electrochemical, photophysical and photochemical properties have been investigated in CH₃CN. The cyclic voltammetry exhibits two successive reversible oxidation processes, corresponding to the Co^III/Co^II and Ru^III/Ru^II redox couples at E_1/2 = −0.06 and 0.91 V vs Ag/Ag⁺ 10 mM, respectively. The one-electron oxidized form of the complex, [{Ru^II(bpy)₂(bpy-terpy)}₂Co^III]⁷⁺ (1⁷⁺) obtained after exhaustive electrolysis carried out at 0.2 V is fully stable. 1⁶⁺ and 1⁷⁺ are only poorly luminescent, indicating that the covalent linkage of the Ru(II)-tris-bipyridine centre to the cobalt subunit leads to a strong quenching of the Ru^II excited state by an intramolecular process. Luminescence lifetime experiments carried out at different temperatures indicate that the transfer is more efficient for 1⁷⁺ compare to 1⁶⁺ due to lower activation energy. Continuous irradiation of 1⁷⁺ performed at 405 nm in the presence of P(Ph)₃ acting as sacrificial electron donor leads to its quantitative reduction into 1⁶⁺, whereas similar experiment starting from 1⁶⁺ with a sulfonium salt as sacrificial electron acceptor converts 1⁶⁺ into 1⁷⁺ with a slower rate and a maximum yield of 80%. These photoinduced electron transfers were followed by UV-Visible spectroscopy and compared with those obtained with a simple mixture of both mononuclear parent complexes i.e. [Ru^II(bpy)₃]²⁺ and [Co^II(tolyl-terpy)₂]²⁺ or [Co^III(tolyl-terpy)₂]³⁺ (tolyl-terpy = 4′-(4-methylphenyl)-2,2′:6′,2′′-terpyridine).     

Sericolophus (Pheronematidae) and Hernandeziana (Corythophoridae) are synonyms (Hexactinellida: Porifera)

Eight specimens of Sericolophus reflexus (Ijima) were compared with the holotype of Hernandeziana ijimae (Hernandez) to assess suspected synonymy of these taxa. An extensive morphometric analysis of the spicules available in these specimens of variable condition show that the H. ijimae holotype exhibits characters near the centre of the range of S. reflexus. It is concluded that H. ijimae is a junior synonym of S. reflexus , and that the family Corythophoridae de Laubenfels is invalid by preoccupation of its base generic name.     

Synthesis and luminescence properties of a blue-emitting Sr(3.5) Y(6.5) O(2) (PO(4) )(1.5) (SiO(4) )(4.5) :Eu(2+) phosphor

A novel blue‐emitting Sr_3.5Y_6.5O₂(PO₄)_1.5(SiO₄)_4.5:Eu²⁺ phosphor was synthesized via a solid‐state reaction. Powder X‐ray diffraction (XRD) analysis demonstrated that the Sr_3.5Y_6.5O₂(PO₄)_1.5(SiO₄)_4.5 host had a hexagonal crystal structure in the space group P6₃/m and unit cell parameters a = 9.418 Å, c = 6.900 Å. The as‐prepared phosphor showed a blue emission and all the main emission peaks were located at around 466 nm for different excitation wavelengths of 297, 333 and 391 nm. The temperature dependence of the photoluminescence property was investigated in the range 20–250 °C, and the emission intensity decreased to 71% of the initial value at room temperature on increasing the temperature to 150 °C. According to the classical theory of fluorescent thermal quenching, the activation energy (ΔE) for the thermal quenching luminescence of the as‐prepared Sr_3.45Y_6.5O₂(PO₄)_1.5(SiO₄)_4.5:0.05Eu²⁺ phosphor was determined to be 0.20 eV. Copyright © 2011 John Wiley & Sons, Ltd.     

The larval head of Exechia (Mycetophilidae) and Bibio (Bibionidae) (Diptera)

Exechia and Bibio have retained several plesiomorphic groundplan features of Diptera and Bibionomorpha, including a fully exposed and sclerotized head capsule, the transverse undivided labrum, the absence of movable premandibles, and undivided mandibles without combs. The fusion of the hypostomal bridge with the head capsule and largely reduced antennae are derived features shared by both taxa. The absence of teeth at the anterior hypostomal margin is a potential autapomorphy of Bibionomorpha. A basal position of Anisopodidae is suggested by a number of plesiomorphies retained in this family. Apomorphies of Bibionomorpha excluding Anisopodidae are the reduction of tentorial elements, the partial fusion of the labrum and clypeus, one-segmented antennae, the absence of a separate submental sclerite, the loss of the labial palpus, and the reduction of the pharyngeal filter apparatus. Head structures of Bibio are largely unmodified. The subprognathous orientation is one of few autapomorphic features. In contrast, the mouthparts of Exechia are highly modified in correlation with the specialized food uptake. The rasping counterrotating movements of maxillae and mandibles with teeth oriented in opposite directions are carried out by strongly developed extensors and flexors of the paired mouthparts. The modified labium mechanically supports the “drill head” formed by the mandibles und maxillae. The necessary stability of the head capsule is provided by the hypostomal bridge which also compensates the far-reaching reduction of the tentorium.     

The helix-coil transitions of poly (dA)-poly (dT) and poly (dA-dT)-poly (dA-dT)

Helix–coil transition curves are calculated for poly (dA) poly(dT) and poly (dA-dT) poly (dA-dT) using the integral equation approach of Goel and Montroll.⁵ The transitions are described by the loop entropy model with the exponent of the loop entropy factor, k, remaining an arbitrary constant. The theoretical calculations are compared with experimental transition curves of the two polymers. Results indicate that the stacking energies for these two polymers differ by about 1 kcal/mole of base pairs. Also, a fit between theory and experiment was not possible for k > 1.70.