滇黄精叶绿体全基因组序列及其密码子使用偏性分析

为探究滇黄精(Polygonatum kingianum)叶绿体全基因组特征和密码子使用偏性,利用第二代测序技术对滇黄精嫩叶进行测序,再经组装与注释后得到其叶绿体基因组全序列,通过MISA、EMBOSS和CodonW等软件对滇黄精叶绿体全基因组的SSR位点、系统发育及密码子偏好性进行分析。结果表明,滇黄精完整叶绿体基因组长度为155 852 bp,基因组平均GC含量为37.7%,其大、小单拷贝区(LSC)长度分别为84 633和185 25 bp,反向重复区长度为26 347 bp,注释了132个基因,包括86个蛋白编码基因、38个tRNA基因和8个核糖rRNA基因。叶绿体基因组中共有69个SSR位点,绝大多数属于单碱基重复的A/T类型。系统发育分析表明滇黄精与格脉黄精(P. tessellatum)亲缘关系近,可能与分布地域有关。密码子偏好性分析表明,滇黄精叶绿体基因组密码子使用模式受到自然选择影响大于突变因素,最终确定9个最优密码子。因此, 滇黄精叶绿体基因组遗传结构和系统发育位置及其密码子偏倚的分析,为叶绿体基因工程研究提供理论依据。     

Chloroplast and mitochondrial DNA diversity in Theobroma cacao

The variability of cocoa (Theobroma cacao) cytoplasmic genomes has been investigated. A total of 177 cocoa clones was surveyed for restriction fragment length polymorphism (RFLP) in chloroplast DNA and in mitochondrial DNA using two restriction endonucleases and various heterologous cytoplasmic probes. A high level of polymorphism was found for the mitochondrial genome. This study points up a structuring of the species that fits with the distinction between the Criollo and Forastero populations. In contrast to all previous analyses, a higher level of polymorphism is found among the Criollo clones while the Forastero clones form quite a homogeneous group.     

菱叶绣线菊彩叶园艺品种‘粉霜’和‘黄金喷泉’的叶绿体基因组研究

基于菱叶绣线菊(Spiraea×vanhouttei)培育出了很多的彩叶园艺品种,其中‘粉霜’彩叶绣线菊(‘Pink Ice’)和‘黄金喷泉’菱叶绣线菊(‘Gold Fountain’)是两个性状优良的品种,这两个彩叶品种的形成机制尚缺乏深入研究。该研究基于二代测序的浅层测序技术,对‘粉霜’和‘黄金喷泉’的叶绿体基因组进行组装、注释、绘制其叶绿体基因组图谱;结合网上已有的绣线菊属植物的叶绿体全基因组开展比较基因组学研究。结果表明,两个品种的叶绿体基因组均为典型的四分体结构,即含有1个LSC、1个SSC及2个IR;‘粉霜’和‘黄金喷泉’的叶绿体基因组大小分别为155 953和155 941 bp,各含有130个基因,包括85个蛋白编码基因,37个转运RNA基因和8个核糖体RNA基因;分别含有67、69个简单重复序列,其中,单核苷酸重复序列最多。筛选出这两个叶绿体基因组内7个高变异区域,分别为trnH_GUG-psbA、trnK_UUU、trnR_UCU-atpA、trnT_ GCU-psbD、ndhC、rpl32、ycf1。菱叶绣线菊、‘粉霜’和‘黄金喷泉’虽有非常近缘的关系,但并未聚成单系。该研究首次获得了两种绣线菊属彩叶品种的叶绿体基因组,为进一步理解绣线菊属及其彩叶园艺品种的亲缘关系提供了大量有用信息,并为今后该属更多园艺资源的发掘奠定了基础。     

Mapping of a chloroplast RFLP marker associated with the CMS cytoplasm of sugar beet (Beta vulgaris)

The Owen cytoplasm of male-sterile sugar beet is associated with several alterations of mitochondrial DNA and one additional HindIII site of chloroplast DNA. The region of this HindIII site has been cloned and sequenced. The site maps in a small reading frame (orf32) close to the ycf7 (orf31) gene in the petG-psbE region of chloroplast DNA. Possible functional implications of the results are discussed. The chloroplast RFLP marker described could be useful for studies on chloroplast-mitochondrial interactions, CMS of sugar beet, and the origin of the Owen cytoplasm.     

The Role of Context-Dependent Mutations in Generating Compositional and Codon Usage Bias in Grass Chloroplast DNA

Abstract The influence of local base composition on mutations in chloroplast DNA (cpDNA) is studied in detail and the resulting, empirically derived, mutation dynamics are used to analyze both base composition and codon usage bias. A 4 × 4 substitution matrix is generated for each of the 16 possible flanking base combinations (contexts) using 17,253 noncoding sites, 1309 of which are variable, from an alignment of three complete grass chloroplast genome sequences. It is shown that substitution bias at these sites is correlated with flanking base composition and that the A+T content of these flanking sites as well as the number of flanking pyrimidines on the same strand appears to have general influences on substitution properties. The context-dependent equilibrium base frequencies predicted from these matrices are then applied to two analyses. The first examines whether or not context dependency of mutations is sufficient to generate average compositional differences between noncoding cpDNA and silent sites of coding sequences. It is found that these two classes of sites exist, on average, in very different contexts and that the observed mutation dynamics are expected to generate significant differences in overall composition bias that are similar to the differences observed in cpDNA. Context dependency, however, cannot account for all of the observed differences: although silent sites in coding regions appear to be at the equilibrium predicted, noncoding cpDNA has a significantly lower A+T content than expected from its own substitution dynamics, possibly due to the influence of indels. The second study examines the codon usage of low-expression chloroplast genes. When context is accounted for, codon usage is very similar to what is predicted by the substitution dynamics of noncoding cpDNA. However, certain codon groups show significant deviation when followed by a purine in a manner suggesting some form of weak selection other than translation efficiency. Overall, the findings indicate that a full understanding of mutational dynamics is critical to understanding the role selection plays in generating composition bias and sequence structure.     

Selection on the codon bias ofChlamydomonas reinhardtii chloroplast genes and the plantpsbA gene

Plant chloroplast genes have a codon use that reflects the genome compositional bias of a high A+T content with the single exception of the highly translatedpsbA gene which codes for the photosystem II D1 protein. The codon usage of plantpsbA corresponds more closely to the limited tRNA population of the chloroplast and is very similar to the codon use observed in the chloroplast genes of the green algaChlamydomonas reinhardtii. This pattern of codon use may be an adaptation for increased translation efficiency. A correspondence between codon use of plantpsbA andChlamydomonas chloroplast genes and the tRNAs coded by the chloroplast genome, however, is not observed in all synonymous codon groups. It is shown here that the degree of correspondence between codon use and tRNA population in different synonymous groups is correlated with the second codon position composition. Synonymous groups with an A or T at the second codon position have a high representation of codons for which a complementary tRNA is coded by the chloroplast genome. Those with a G or C at the second position have an increased representation of codons that bind a chloroplast tRNA by wobble. It is proposed that the difference between synonymous groups in terms of codon adaptation to the tRNA population in plantpsbA andChlamydomonas chloroplast genes may be the result of differences in second position composition.     

Comparison of dinucleotide frequency and codon usage inToxoplasma andPlasmodium: Evolutionary implications

Summary The weight-averaged observed/expected dinucleotide frequencies for the sum total of the coding regions of fiveToxoplasma genes were compared with the same parameters previously determined for the coding regions of 21Plasmodium genes. In addition, codon usage in the fiveToxoplasma genes was compared with that in the 21Plasmodium genes, and the percent distribution of amino acids in theToxoplasma protein pool and thePlasmodium protein pool were compared with that in a general protein pool of 314 proteins. The results are consistent with the hypothesis that, contrary to currently held opinion, the generaToxoplasma andPlasmodium are not especially closely related.     

4种霸王属植物叶绿体基因组密码子偏好性分析

【目的】确定4种霸王属植物叶绿体基因组密码子的使用模式及其成因。【方法】利用Codon W、Emboss等对叶绿体基因组筛选出40条基因编码序列密码进行ENC-plot、中性绘图、最优密码子等分析。【结果】4种霸王属植物叶绿体基因组密码子的GC含量依次为GC1>GC2>GC3,ENC值平均值都大于35,CAI均为0.17,其基因表达水平较低,且密码子使用偏好性较弱;中性绘图分析、ENC-plot及PR2-plot分析认为4个物种叶绿体基因组密码子的偏好性主要受自然选择作用的影响;最优密码子分析发现豆型霸王、长梗霸王、霸王和喀什霸王最优密码子分别为19、13、14和14个,其中4个为共有密码子且偏好以A/U收尾。【结论】本研究为4种霸王属植物的密码子碱基多以AU结尾,偏好性较弱且主要影响因素为自然选择,鉴定了4个共有最优密码子;这对于深入理解霸王属植物的密码子优化、偏好机制研究及遗传进化关系提供科学依据。     

白鲜叶绿体高通量测序组装及系统进化分析

通过高通量测序技术获取白鲜（Dictamnus dasycarpus Turcz.）叶绿体全基因组序列,并采用生物信息学方法对其结构特征和系统发育关系进行了分析。结果显示,白鲜叶绿体基因组具有典型的环状四分体结构,总长度为157 139 bp,大单拷贝区和小单拷贝区长度分别为84 478 bp和18 587 bp,一对反向重复序列长度为27 037 bp,总GC含量为38.5%。共注释到132个基因,包括87个蛋白编码基因（PCGs）、8个rRNA和37个tRNA基因。计算相对同义密码子使用频率（RSCU）,发现编码最多的密码子是亮氨酸（Leu/L）,编码最少的密码子是半胱氨酸（Cys/C）,RSCU值大于1的密码子有32个。白鲜叶绿体基因组共包含61个简单重复序列。通过对芸香科8种植物的叶绿体基因组结构进行比较,发现白鲜属与其他属的叶绿体基因组结构基本一致,并未发生重排和倒位现象。系统进化树分析结果表明,白鲜与臭常山（Orixa japonica Thunb.）的亲缘关系最近。     

Light-dependent particle movement in the outer envelope of chloroplasts fromCodium australicum (Silva)

Summary Chloroplasts from light treatedCodium australicum (Silva) show two regions of differing particle density in the outer envelope EF face, both in fixed and unfixed material. The region with high particle density is always oriented toward the cell wall. Dark treated chloroplasts show no such division. Particle size histograms indicate that this division is chiefly the result of a shift of existing large particles toward the cell wall in the light. Some addition of new material may also occur.     

Synthesis of Drosophila melanogaster acetylcholinesterase gene using yeast preferred codons and its expression in Pichia pastoris

To improve the expression level of recombinant Drosophila melanogaster AChE (R-DmAChE) in Pichia pastoris, the cDNA of DmAChE was first optimized and synthesized based on the preferred codon usage of P. pastoris. The synthesized AChE cDNA without glycosylphosphatidylinositol (GPI) signal peptide sequence was then ligated to the P. pastoris expression vector, generating the plasmid pPIC9K/DmAChE. The linearized plasmid was homologously integrated into the genome of P. pastoris GS115 via electrotransformation. Finally seven transformants with high expression level of R-DmAChE activity were obtained. The highest production of R-DmAChE in shake-flask culture after 5-day induction by methanol was 718.50 units/mL, which was about three times higher than our previous expression level of native DmAChE gene in P. pastoris. Thus, these new strains with the ability to secret R-DmAChE in the medium could be used for production of R-DmAChE to decrease the cost of the enzyme expense for rapid detection of organophosphate and carbamate insecticide residues.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

滇南青冈叶绿体基因组特征及密码子使用偏好性分析

为了明确滇南青冈（Quercus austroglauca Y. T. Chang）叶绿体基因组的结构特征、密码子使用偏好性及其影响因素,本研究运用Geneious、CodonW等软件,对其进行了系统分析。结果显示,滇南青冈的叶绿体基因组全长160 913 bp,编码133个基因。从中筛选出的52条蛋白编码序列第3位碱基的平均GC含量为29.14%,说明其密码子偏爱以A或U结尾,相对同义密码子使用度分析结果也印证了这一点。有效密码子数（ENC）均大于35,表明其密码子偏好性较弱。中性绘图分析、ENC-plot分析以及PR2-plot分析结果均表明,影响滇南青冈叶绿体基因组密码子使用偏好性的主导因素是自然选择。通过筛选确定,其最优密码子共有13个。多重分析结果显示,变异多在非编码区域,编码区ycf1基因的变异程度较大。IR边界分析显示滇南青冈存在ycf1假基因。系统发育分析表明滇南青冈与多脉青冈（Q. hypargyrea (Seemen) C. C. Huang et Y. T. Chang）等栎属植物亲缘关系较近。     

Time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas reinhardii

Contradictory data have been published concerning the time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas. In the present study, adjacent ultrathin sections of Chlamydomonas reinhardii zygotes of various ages were examined with the electron microscope. These sections clearly reveal that nuclear fusion precedes chloroplast fusion.     

Chloroplast DNA restriction site mapping and the phylogeny ofRanunculus (Ranunculaceae)

A chloroplast DNA restriction site map forRanunculus sceleratus (Ranunculaceae) was constructed using 14 restriction endonucleases. The total size of the chloroplast genome is 152.4kb. No inversions were detected relative to the tobacco chloroplast DNA. Cladistic analyses of chloroplast DNA restriction site polymorphism were employed in order to elucidate the phylogeny among 76 species of the genusRanunculus in a wide sense and one species ofTrautvetteria. A total of 341 informative restriction site changes were detected. Parsimony jackknifing, bootstrapping and decay analysis were undertaken in order to evaluate the amount of support for the monophyletic groups. The results suggest that the analysed species ofRanunculus are divisible into two main clades. Only few of the traditional sections and subgenera ofRanunculus are monophyletic. The genusTrautvetteria is nested within a clade comprising, e.g.Ranunculus cymbalaria, R. andersonii, R. lapponicus andR. ficaria. SubgenusBatrachium lies within a larger clade containing, e.g.R. sceleratus andR. hyperboreus. Contractions of the inverted repeat due to parallel deletions of 200–300 bp close to the J_SB have occurred in many clades and the phylogenetic distribution of this size reduction was mapped among the species.     

TheUlmaceae, one family or two? Evidence from chloroplast DNA restriction site mapping

The Ulmaceae is usually split into two subgroups, referred to as either tribes or more commonly subfamilies (Ulmoideae andCeltidoideae). The two groups are separated, with some exceptions, on the basis of leaf venation, fruit type, seed morphology, wood anatomy, palynology, chemistry, and chromosome number. Propositions to separate the two groups as distinct families have never gained general acceptance. Recent morphological and anatomical data have suggested, however, that not only is family status warranted but thatCeltidaceae are more closely related toMoraceae and otherUrticales than toUlmaceae. In order to test these alternative sets of relationships, restriction site mapping of the entire cpDNA was done with nine rare cutting enzymes using 11 genera ofUlmaceae s. l., three other families of theUrticales, and an outgroup family from theHamamelidae. Cladistic analysis of the data indicates thatUlmaceae s. l. is not monophyletic and that distinct families (Ulmaceae andCeltidaceae) are warranted; thatUlmaceae is the sister group toCeltidaceae plus all other families in the order; and thatCannabaceae might be nested withinCeltidaceae. Familial placements of various problematic genera (e.g.Ampelocera, Aphananthe) are resolved and character evolution of key morphological, anatomical, chemical, and chromosomal features are discussed.