Construction and characterisation of a yeast artificial chromosome library containing three haploid maize genome equivalents

We have constructed a yeast artificial chromosome (YAC) library using high-molecular-weight DNA prepared from agarose-embedded leaf protoplasts of the maize inbred line UE95. This library contains 79 000 clones with an average insert size of 145 kb and should therefore represent approximately three haploid genome equivalents. The library is organised as an ordered array in duplicate microtitre plates. Forty-one pools of DNA from 1920 individual clones have been prepared for rapid screening of the library by the polymerase chain reaction (PCR). Using this approach, together with conventional colony hybridisation, we have been able to identify between one and eight positive clones for every probe used.     

猪圆环病毒2型流行株的分离及其感染性克隆的构建

【目的】通过分离一株猪圆环病毒2型(PCV2)流行毒株,并构建其感染性克隆,为研究PCV2基因功能提供操作平台。【方法】通过PCR方法,从疑似患断奶仔猪多系统衰竭综合症(PMWS)的仔猪淋巴结中鉴定为猪圆环病毒(Porcine circovirus,PCV)2型阳性。把阳性病料接种PK-15细胞传代培养,在培养物中扩增出PCV2的全基因序列。对扩增出的全序列进行序列测定,并与GenBank中公布的5株广东PCV2分离株(GD-pz、GD-gj、GD-jm、GD-ss和GD-sz)进行同源性分析。通过EcoRⅠ和SalⅠ将PCV2全基因组序列克隆进pUC18载体中,获得含PCV2 GD-zq株全基因组单拷贝的重组质粒pPCV2-GD-zq,再通过SalⅠ和HindⅢ把另一个全长拷贝克隆进pPCV2-GD-zq质粒中,使PCV2 GD-zq株基因组DNA以头尾相接的双重复方式克隆进pUC18载体中,获得重组质粒pPCV2-2GD-zq。将pPCV2-2GD-zq DNA纯化和定量后转染PK-15细胞,拯救PCV2 GD-zq病毒。【结果】从PMWS感染的猪淋巴结中分离到了一株PCV2,命名为GD-zq株;序列分析结果显示,GD-zq株全基因组为1 767 bp,与GenBank中公布的5株广东PCV2分离株ORF1核苷酸一致性为97.1%-99.7%,编码氨基酸一致性为98.7%-100%;ORF2核苷酸一致性为93.2%-99.6%,编码氨基酸一致性为92.3%-99.1%;全基因一致性为96.0%-99.6%。pPCV2-2GD-zq质粒转染PK-15细胞后,其通过间接免疫荧光实验(IFA)能从转染细胞及其传代细胞中,检测到拯救出的病毒。【结论】分离了一株PCV2广东株GD-zq,成功构建了PCV2 GD-zq株的感染性克隆。     

Chromosome-level genome assembly and characterization of Sophora Japonica

Sophora japonica is a medium-size deciduous tree belonging to Leguminosae family and famous for its high ecological, economic and medicinal value. Here, we reveal a draft genome of S. japonica, which was ∼511.49 Mb long (contig N50 size of 17.34 Mb) based on Illumina, Nanopore and Hi-C data. We reliably assembled 110 contigs into 14 chromosomes, representing 91.62% of the total genome, with an improved N50 size of 31.32 Mb based on Hi-C data. Further investigation identified 271.76 Mb (53.13%) of repetitive sequences and 31,000 protein-coding genes, of which 30,721 (99.1%) were functionally annotated. Phylogenetic analysis indicates that S. japonica separated from Arabidopsis thaliana and Glycine max ∼107.53 and 61.24 million years ago, respectively. We detected evidence of species-specific and common-legume whole-genome duplication events in S. japonica. We further found that multiple TF families (e.g. BBX and PAL) have expanded in S. japonica, which might have led to its enhanced tolerance to abiotic stress. In addition, S. japonica harbours more genes involved in the lignin and cellulose biosynthesis pathways than the other two species. Finally, population genomic analyses revealed no obvious differentiation among geographical groups and the effective population size continuously declined since 2 Ma. Our genomic data provide a powerful comparative framework to study the adaptation, evolution and active ingredients biosynthesis in S. japonica. More importantly, our high-quality S. japonica genome is important for elucidating the biosynthesis of its main bioactive components, and improving its production and/or processing.     

蒙古狼线粒体基因组序列分析及其系统发育地位

应用Long-PCR和克隆测序法得到蒙古狼(Canis lupus chanco)线粒体基因组全序列,结合GenBank中现有犬科动物线粒体基因组数据,应用最大简约法(MP)、最大似然法(ML)和Bayesian分析法对蒙古狼的系统发育地位进行了探讨。结果如下:蒙古狼线粒体基因组全长16709bp,包含13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和1个非编码区。序列碱基的组成存在明显的A-T偏好性。tRNA基因中除tRNA-Ser(AGY)缺少双氢尿嘧啶(DHU)臂以外,其余均能折叠成典型的三叶草二级结构。大多数蛋白质编码基因的起始和终止密码子与犬科动物有报道相同,COXⅡ基因的起始密码子为ATA,与其他犬科动物不同。基于12S rRNA+16S rRNA+H链上的12个蛋白质编码基因的联合数据的系统发育分析发现,在已报道的狼亚种数据中,西藏狼(Canis lupus laniger)的分化时间最早,其次为阿拉伯狼(Canis lupus arabs),蒙古狼与欧亚狼(Canis lupuslupus)的系统发育地位最为接近。     

毛茛科和芍药科叶绿体基因组密码子特征和系统发育比较

为研究和比较毛茛科和芍药科叶绿体基因组密码子使用模式和系统进化关系,以完成测序的毛茛科33种植物、芍药科7种植物叶绿体基因组为材料,采用分析软件CodonW在线软件CUSP和R软件对叶绿体基因进行密码子特征分析。用MAFFT软件,MEGA软件进行系统发育分析。研究结果表明芍药科植物叶绿体基因组和毛茛科植物（耧斗菜属除外）叶绿体基因组高频密码子一致性高,具有29个高频密码子,基本偏向与于A/U结尾,但最优密码子存在差异。毛茛科和芍药科叶绿体基因组密码子偏好性的形成因素主要受自然选择的影响,且芍药科叶绿体基因组密码子偏好性受自然选择的影响大于毛茛科。基于叶绿体基因组全序列和基于叶绿体基因组CDS序列的系统进化关系表明,芍药科基于叶绿体基因组全序列和基于叶绿体基因组CDS序列的系统进化关系虽然部分不同,但都可以被划分为芍药组和牡丹组。毛茛科基于叶绿体基因组的系统进化关系不符合中国植物志分类关系,但支持把毛茛科划分为4亚科14族。系统进化分析结果也支持芍药科独立于毛茛科和毛茛目,被划分到虎耳草目,同时证明了叶绿体基因组作为超级DNA条形码的可行性。     

快速获取55型腺病毒基因组序列的方法

【目的】建立快速获取55型腺病毒的全长基因组序列的方法。【方法】根据55型腺病毒的基因组特点,设计覆盖55型腺病毒基因组序列的12对引物,分别以55型腺病毒DNA为模板,扩增得到12个PCR产物,通过对12个PCR产物测序及序列拼接,获得55型腺病毒的全长基因组序列。【结果】从本院急性上呼吸道感染者咽拭子标本中分离得到一株55型腺病毒毒株SF04/SC/2016,以其DNA为模板扩增成功获得12个PCR产物,对其进行测序,并对12段序列进行拼接得到55型腺病毒的全长基因组序列,与已报到的各型腺病毒序列进行比对,采用邻位相连法构建系统发育进化树,所得序列与55型腺病毒处于同一分支,进一步确认该病原体为55型腺病毒。【结论】研究公布的序列和方法,能够实现更方便对腺病毒的快速测序,为揭示55型腺病毒的进化特点及制订疾病防控策略提供了有效手段。     

近缘细菌细胞间的相互识别与相互作用

亲缘识别是细菌细胞间竞争与合作的前提和基础。细菌通过亲缘识别分辨自我细胞和非自我细胞;非同类的细菌细胞相互分离或被排除,而亲缘种群内的细菌细胞进行群体运动、生物膜和子实体形成等社会性合作行为。细菌的自我识别机制可能有助于不同亲缘类群在混杂的自然系统中的共存。近些年来,细菌亲缘识别及相互作用的机制研究工作成为热点,本文总结了近缘细菌细胞间相互识别和作用机制的研究进展。