兴国红鲤MHCⅡ类α基因多态性、表达及其与鱼体抗病力关系的分析

对41尾感病和22尾抗病兴国红鲤的308个有效克隆进行测序,获得171条不同的MHCⅡ类α基因编码序列,分属26个不同的等位基因,其中Cyca-DXA24—Cyca-DXA36为新发现的13个等位基因。MHCⅡ类α基因片段的长度为624 bp,包括第1—4个外显子,分别编码信号肽、α1和α2结构域及连接肽/跨膜区。α1结构域的变异明显大于α2结构域,表现在α1结构域中核苷酸和氨基酸变异位点比例(55.16%和79.76%)明显高于α2结构域的变异位点比例(45.96%和68.42%)。α1结构域的PBR区的非同义碱基替换率(dN)与同义碱基替换率(dS)的比值ω(ω=dN/dS)为5.742,远远高于非抗原结合位点(non-PBR)及α2结构域的0.755、0.592,揭示兴国红鲤MHCⅡ类α基因的α1结构域在进化过程中受到正向选择作用。等位基因Cyca-DXA24(P0.01)与兴国红鲤对嗜水气单胞菌的抗性相关,等位基因Cyca-DXA3(P0.05)、Cyca-DXA4(P0.01)、Cyca-DXA6(P0.05)、Cyca-DXA33(P0.05)与兴国红鲤对嗜水气单胞菌的易感性相关。荧光定量PCR结果表明,MHCⅡ类α基因在健康兴国红鲤的肾、肝、鳃等10个组织均能普遍表达。人工感染嗜水气单胞菌后,肾、肝、脾3个组织中的MHCⅡ类α基因的表达量均发生了不同程度的变化,表明MHCⅡ类α分子在兴国红鲤的免疫反应中起到重要作用。     

乌龟MHCⅡ类B基因第二外显子的克隆及序列分析

利用一对兼并性引物扩增了乌龟MHCⅡ类分子B基因第二外显子的部分片段,并对PCR产物进行了克隆和测序,结果得到8种长度为166 bp的不同序列。经分析,序列中有84个变异位点,核苷酸的非同义替换(dN)多于同义替换(dS),造成39个位点氨基酸的改变。氨基酸的替换趋于集中在假定的抗原结合位点附近。利用MEGA、PAUP软件分别构建NJ树和MP树,两种树极为相似,均分为两支。同一个体中出现有多种序列,提示乌龟MHCⅡ类分子B基因可能存在着座位重复。研究表明:乌龟MHCⅡ类分子B基因第二外显子有较高的多态性,有利于乌龟野生种群的遗传保护。     

扬子鳄MHCⅡ类B基因第二外元的克隆及序列分析

3头扬子鳄血样取自宣城安徽省扬子鳄繁殖研究中心。利用一对简并引物对MHCⅡ类B基因第二外元的部分片段进行扩增;通过克隆、单链构象多态性分析、测序,并将测得序列与下载的8个物种MHC序列比对,确定序列差异和变异位点;利用MEGA软件构建NJ树,PAUP4．0构建MP树。结果得到10种不同的序列,片段长166bp。核苷酸序列中有38个变异位点,氨基酸序列中有23个变异位点;推定的抗原结合位点非同义替换(dN)明显高于同义替换(dS)。10种序列的NJ树和MP树极为相似,均为A、B两个分支,两个分支明显的特异性位点核苷酸序列中有9个。氨基酸序列中有7个。表明扬子鳄MHCⅡ类B基因第二外元有较高的多态性,有利于扬子鳄饲养种群的遗传保护。     

中国红鲤线粒体COⅡ基因的遗传变异和亲缘关系

通过线粒体COⅡ基因的序列分析,研究了我国4种红鲤——兴国红鲤(C．c．var．singuonensis)、荷包红鲤(C．c．var．wuyuanensis)、玻璃红鲤(C．c．var．wananensis)、瓯江彩鲤(C．c．var．color)和1种野鲤(Cyprinus carpio)的遗传变异和亲缘关系。结果表明：在所分析的607bp片段中,共有19个核苷酸变异位点,可归结为15种基因单倍型,其中5个变异位点可作为群体鉴别的单核苷酸标记(SNP);荷包红鲤的基因多样性(h)和核苷酸多样性(π)最高,玻璃红鲤最低;所有红鲤均存在极显著的遗传分化,其中兴国红鲤与玻璃红鲤的遗传分化指数(Fst)最高,而兴国红鲤与瓯江彩鲤的遗传分化指数最低。用邻接法构建的分子系统树表明,兴国红鲤与瓯江彩鲤属同一进化分支,而玻璃红鲤与荷包红鲤属另一进化分支。     

嗜水气单胞菌丝氨酸蛋白酶基因克隆与序列分析

目的:克隆嗜水气单胞菌丝氨酸蛋白酶(Ahp)基因,进一步分析序列、预测三级结构。方法:设计Ahp基因特异性引物,利用PCR方法扩增基因序列,亚克隆至pMD18-T载体,进行DNA测序及生物信息学分析。结果:获得Ahp基因大小为1 645bp,推导编码548aa,含有天冬氨酸(82-93aa)、组氨酸(123-133aa)和丝氨酸活性位点(342-352aa)。Ahp蛋白三级结构与模板(PDB:3HJR_A)相似性达81.48%,预测60-392aa区域为肽酶S8结构域(PF00082)、480-548aa区域为前蛋白转化酶P结构域(PF01483),其中3个氨基酸活性位点分布于三级结构N端,与拉马钱德兰图检测分析Ahp蛋白的空间结构基本一致。结论:该研究对嗜水气单胞菌Zf1菌株Ahp基因编码蛋白进行结构预测,有助于理解嗜水气单胞菌丝氨酸蛋白酶的作用机理。     

中国地方鸡种MHC B-LBⅡ新等位基因的遗传多态性研究

为研究鸡MHC B-LBⅡ基因的遗传多态性,首先在8个中国地方鸡种(藏鸡、仙居鸡、北京油鸡、固始鸡、斗鸡、丝羽乌骨鸡、白耳鸡和狼山鸡)B-LBⅡ基因第二外显子扩增了一长度为 175 bp 的 DNA 片段并进行 SSCP 基因型分析;在8 个地方鸡种共 467 个个体中检测到 37 个 PCR-SSCP 基因型;从被检样品中筛选出不同基因型的个体,并在其 B-LBⅡ基因组中扩增了一个包括其第二外显子和第二内含子在内长度为374 bp的片段,通过克隆和测序获得了该片段的核苷酸序列。经序列分析,在前述地方鸡种被筛选出的 30 个无血缘关系的个体中发现了 31 个 B-LBⅡ新等位基因,并参照哺乳动物 MHC II 类 B 等位基因命名规则进行了命名。对这 31 个 B-LBⅡ新等位基因长度为 374 bp 的 DNA 片段进行比对表明,在其第二外显子序列上共有 68 个多态性变异位点,其中简约性信息位点 51 个,单变异位点 17 个,具有丰富的遗传多态性。在这些多态性变异位点中,出现在遗传密码子第一和第二位上的碱基替换率分别为 36.76% 和 35.29%。等位基因序列间的相似性估测为 90.6%-99.5%;B-LBⅡ基因第二外显子的错义替换率和同义替换率分别为 14.64±2.67%和 2.92±0.94%。结果表明,B-LBⅡ基因的丰富遗传多态性主要是由基因重组和平衡选择效应所引起的。对 B-LBⅡ等位基因第二外显子所编码的 B-LBⅡ分子β1 结构域氨基酸序列比对发现,31 个 B-LBⅡ新等位基因属于 26 个等位基因主型;在β1结构域氨基酸序列的 33个变异位点上,存在 6 个同义替换和 27 个错义替换。分析认为,那些发生在多肽结合位点上的氨基酸错义替换与鸡 MHC B-LBⅡ分子的免疫特异性有关。该结果可为鸡的抗病育种研究提供分子生物学依据。     

嗜水气单胞菌胁迫下东北林蛙MHCⅠ基因在不同组织的表达

本研究旨在探讨嗜水气单胞菌(Aeromonashydrophila,Ah)胁迫下MHCⅠ基因在东北林蛙不同组织的表达特征,为揭示两栖类抗感染免疫应答机制提供依据。文中首先构建嗜水气单胞菌感染下的试验动物模型,通过苏木精-伊红染色法(Hematoxylin-eosin staining,HE染色)观察病理学变化;利用RT-PCR克隆东北林蛙MHCⅠ基因α1+α2肽结合区并进行生物信息学分析,再运用荧光定量PCR技术检测Ah胁迫下MHCⅠ在不同组织中的转录水平。结果表明:在Ah感染后,皮肤、肝脏和肌肉等组织均出现细胞结构消失和纹理紊乱等现象;获得MHCⅠ基因α1+α2肽结合区片段494bp,可编码164个氨基酸,与两栖类同源性在77%以上,与哺乳类同源性低至14.96%,表明MHCⅠ基因α1+α2区在不同物种间保守性较低;荧光定量PCR结果显示,在Ah胁迫下,实验组肝脏、脾脏、肾脏、皮肤和肌肉组织MHCⅠ基因的转录水平在72 h前均高于对照组,但是各组织到达峰值的时间具有差异性(P0.01),表明Ah胁迫下MHCⅠ基因在不同组织的表达时间存在差异。研究结果为进一步探究MHC分子在抗感染中的免疫功能提供了参考依据。     

凹耳蛙MHCII类B基因第二外显子多态性分析

两栖类正经历全球范围内的种群衰退,很多两栖动物集群灭绝事件与环境病原体(如壶菌(Batrachochytrium dendrobatidis)的侵扰有关。MHC基因的表达产物在有颌脊椎动物免疫应答过程中起关键作用,其多态性通常与动物对疾病的抗性或易感性密切相关,因而被认为是研究动物适应性进化的最佳候选基因之一。本文对中国特有的无尾两栖动物凹耳蛙(Odorrana tormota)MHC II类B基因多态性进行初步研究。首先,利用1对通用引物扩增出凹耳蛙MHC II类B基因exon2长约180bp的DNA片段。在此基础上,利用ligation-mediated PCR进一步获取侧翼未知序列,序列拼接后长2,030bp,包含exon2以及intron1和intron2的部分序列。基于上述序列设计出凹耳蛙B基因exon2特异性引物(IIQ1BU/IIQ1BD),对该物种黄山种群32个样品进行PCR扩增和克隆测序,共获得34个不同的等位基因,等位基因序列核苷酸和氨基酸变异位点的比例分别为16.17%(33/204)和26.87%(18/67),大多数氨基酸变异位点位于推测的抗原结合位点(antigen binding sites,ABS)。每个样品包含2-5个等位基因,结合等位基因序列特征以及cDNA表达分析结果,推测凹耳蛙至少拥有3个可表达的B基因座位。与文献报道的蛙科其他物种比较后发现,尽管凹耳蛙目前的分布区非常狭窄,但其MHC II类B基因多态性明显高于蛙科其他动物。等位基因碱基替换模式提示凹耳蛙MHC II类B基因曾经历过强烈的正选择作用,ABS区的dN值显著大于dS(P<0.05),PAML软件包CODEML程序中不同模型的似然比检测(likelihood rate test)结果同样支持上述推论,贝叶斯经验贝叶斯路径(Bayesian Em-pirical Bayes)共检测出5个显著受正选择作用的氨基酸位点。贝叶斯系统树的拓扑结构显示,无尾两栖类不同科的等位基因分别形成单系群,但蛙科不同属的等位基因未能形成单系群,蛙属绿池蛙(Rana clamitans)的1个等位基因与臭蛙属凹耳蛙的部分等位基因享有共同的谱系关系,提示蛙科不同属间的B基因存在跨种多态性。     

尼罗鳄MHCⅡ类B基因第二外元的序列变异分析

利用一对简并引物扩增了尼罗鳄MHCⅡ类分子B基因第二外元的部分片段,并对PCR产物进行了克隆和测序,结果得到8种不同的序列,序列长度为166 bp;经分析,序列中有56个变异位点,核苷酸的非同义替换多于同义替换,造成30个位点氨基酸的改变,氨基酸的替换趋于集中在假定的抗原结合位点附近.核苷酸和氨基酸序列与已报道的扬子鳄和密河鳄的MHCⅡ类B基因第二外元序列有较高的同源性,利用PAUP4.0软件构建的NJ树显示,鳄类的MHCⅡ类B基因存在跨种多态性现象.     

嗜水气单胞菌转录调控蛋白基因的克隆与序列分析

根据嗜水气单胞菌(Aeromonas hydrophila)转录调控蛋白(AhyR)基因序列设计特异性引物,采用PCR方法克隆嗜水气单胞菌Zf1菌株AhyR基因,测序目的基因大小为1 063 bp。对AhyR基因编码蛋白进行生物信息学分析,推导其开放阅读框(ORF)编码260 aa,含有自体诱导物结合域(18-168 aa)、LuxR型螺旋-转角-螺旋(HTH)DNA结合结构域(176-241 aa),以及13个磷酸化位点(15S、30T、62S、144S、145S、156S、161Y、173S、184T、188T、200S、227S、231Y)。AhyR蛋白三级结构与模板(PDB:3SZT_A)相似性达28.40%,结果显示LuxR型螺旋-转角-螺旋结构域主要以α螺旋分布在C端外侧,这与拉马钱德兰图(Ramach andran plot)检测分析AhyR蛋白空间结构基本一致。     

At least two regions of the oncoprotein Tre2 are involved in its lack of GAP activity

The product of the human Tre2 oncogene is structurally related to the Ypt/Rab GTPase-activating proteins (Ypt/Rab GAPs). Particularly, the oncoprotein shares with the yeast proteins Msb3p and Msb4p, and with the human protein RN-tre the highly conserved TBC domain, forming the catalytically active domain of Ypt/Rab GAPs. Yet, the Tre2 oncogene seems to encode a nonfunctional Rab GAP. As regions flanking the TBC domain may be crucial for catalytic activity, regions located N- and C-terminally with respect to this domain were explored. For this, chimeric proteins created by sequence exchanges between the Tre2 oncoprotein and RN-tre were tested for their ability to replace functionally the Msb3p and Msb4p proteins in double-mutant yeast cells. These complementation experiments revealed, in addition to the TBC domain, a second Tre2 region involved in the oncoprotein's lack of GAP activity: a 93-aa region flanking the TBC domain on the C-terminal side.     

Identification of nucleopolyhedrovirus that infect Nymphalid butterflies Agraulis vanillae and Dione juno

Dione juno and Agraulis vanillae are very common butterflies in natural gardens in South America, and also bred worldwide. In addition, larvae of these butterflies are considered as pests in crops of Passiflora spp. For these reasons, it is important to identify and describe pathogens of these species, both for preservation purposes and for use in pest control. Baculoviridae is a family of insect viruses that predominantly infect species of Lepidoptera and are used as bioinsecticides. Larvae of D. juno and A. vanillae exhibiting symptoms of baculovirus infection were examined for the presence of baculoviruses by PCR and transmission electron microscopy. Degenerate primers were designed and used to amplify partial sequences from the baculovirus p74, cathepsin, and chitinase genes, along with previously designed primers for amplification of lef-8, lef-9, and polh. Sequence data from these six loci, along with ultrastructural observations on occlusion bodies isolated from the larvae, confirmed that the larvae were infected with nucleopolyhedroviruses from genus Alphabaculovirus. The NPVs from the two different larval hosts appear to be variants of the same, previously undescribed baculovirus species. Phylogenetic analysis of the sequence data placed these NPVs in Alphabaculovirus group I/clade 1b.     

Identification key for European strand-forming house-rot fungi

An identification key for 20 common strand-forming indoor wood decay fungi is given. The key is based on observations of material from affected buildings and on wood samples that have been incubated in the laboratory. The key is with macro- and microscopic photographs.     

Alterations of rx1 and pax6 expression levels at neural plate stages differentially affect the production of retinal cell types and maintenance of retinal stem cell qualities

rx1 and pax6 are necessary for the establishment of the vertebrate eye field and for the maintenance of the retinal stem cells that give rise to multiple retinal cell types. They also are differentially expressed in cellular layers in the retina when cell fates are being specified, and their expression levels differentially affect the production of amacrine cell subtypes. To determine whether rx1 and pax6 expression after the eye field is established simply maintains stem cell-like qualities or affects cell type differentiation, we used hormone-inducible constructs to increase or decrease levels/activity of each protein at two different neural plate stages. Our results indicate that rx1 regulates the size of the retinal stem cell pool because it broadly affected all cell types, whereas pax6 regulates more restricted retinal progenitor cells because it selectively affected different cell types in a time-dependent manner. Analysis of rx1 and pax6 effects on proliferation, and expression of stem cell or differentiation markers demonstrates that rx1 maintains cells in a stem cell state by promoting proliferation and delaying expression of neural identity and differentiation markers. Although pax6 also promotes proliferation, it differentially regulates neural identity and differentiation genes. Thus, these two genes work in parallel to regulate different, but overlapping aspects of retinal cell fate determination.     

Parasites, emerging disease and wildlife conservation

In this review some emerging issues of parasite infections in wildlife, particularly in Australia, are considered. We discuss the importance of understanding parasite biodiversity in wildlife in terms of conservation, the role of wildlife as reservoirs of parasite infection, and the role of parasites within the broader context of the ecosystem. Using a number of parasite species, the value of undertaking longitudinal surveillance in natural systems using non-invasive sampling and molecular tools to characterise infectious agents is illustrated in terms of wildlife health, parasite biodiversity and ecology.     

Phylogenetic relationships among Southeast Asian climbing bamboos (Poaceae: Bambusoideae) and the Bambusa complex

A phylogenetic analysis of Bambusa and allies based on the plastid DNA non-coding regions rps16-trnQ, trnC-rpoB, trnH-psbA and trnD-T, and a partial nuclear GBSSI gene, was carried out. This included representatives from all four Bambusa subgenera (including type species), a group of segregate Southeast Asian genera distinctive by their climbing–scrambling culms (Dinochloa, Holttumochloa, Kinabaluchloa, Maclurochloa, Soejatmia, Sphaerobambos), and two other Bambusinae genera (Dendrocalamus, Gigantochloa). The results do not support the present subgeneric classification of Bambusa. The climbing Southeast Asian genera, all of which include species previously placed in Bambusa, are distinct from the “core Bambusa group” (type species and alliance) and the Bambusa complex generally.     

Co-option of an anteroposterior head axis patterning system for proximodistal patterning of appendages in early bilaterian evolution

The enormous diversity of extant animal forms is a testament to the power of evolution, and much of this diversity has been achieved through the emergence of novel morphological traits. The origin of novel morphological traits is an extremely important issue in biology, and a frequent source of this novelty is co-option of pre-existing genetic systems for new purposes (Carroll et al., 2008). Appendages, such as limbs, fins and antennae, are structures common to many animal body plans which must have arisen at least once, and probably multiple times, in lineages which lacked appendages. We provide evidence that appendage proximodistal patterning genes are expressed in similar registers in the anterior embryonic neurectoderm of Drosophila melanogaster and Saccoglossus kowalevskii (a hemichordate). These results, in concert with existing expression data from a variety of other animals suggest that a pre-existing genetic system for anteroposterior head patterning was co-opted for patterning of the proximodistal axis of appendages of bilaterian animals.