果树叶绿体基因组测序及系统发育研究进展

叶绿体是植物细胞中进行光合作用的细胞器,含有相对独立的基因组信息,这些遗传信息在揭示物种起源、进化及不同物种间的亲缘关系等方面具有重要价值。同时,叶绿体转基因技术因其定点整合、高效表达及安全性好的优势成为植物基因工程的研究热点之一。本文归纳了果树叶绿体分离、叶绿体DNA提取以及测序的发展历程,基于叶绿体全基因组测序结果探讨了果树叶绿体基因组的组成、结构特点及其与系统发育的关系,旨在进一步了解果树叶绿体基因组的研究现状、发展趋势和应用前景。     

中国科学家成功揭示鸡形目所属生物系统发育关系

鸡形目在生物分类学上是鸟纲中的一个目。由于它们具有重要的科学与经济价值,研究倍受学界关注。然而到目前为止,它们的系统发育关系仍然混淆不清,尤其是雉科物种的系统发育关系十分复杂。     

我国南方野生樱属植物的分子系统发育分析

为鉴定我国樱属植物的系统发育关系,本研究以30种典型樱属植物为试验材料,利用核基因ITS和4个叶绿体间隔序列[petA-psbJ、trnH-psbA、rpl32-trnL(UAG)和trnL-trnF]进行测序,采用MEGA 6软件对5个序列特点进行分析,并构建系统发育树。结果表明,5个序列的DNA片段长度多态性和单核苷酸多态性较高,其中ITS和rpl32-trnL(UAG)变异程度最大,可作为樱属植物核心条形码序列,其他序列可作为辅助序列;通过5个序列的组合序列构建的系统发育树,可相对准确地反映樱属植物间的系统发育关系。本研究结果为进一步探究樱属植物的起源、系统发育关系、物种鉴定及资源保护提供了一定的参考依据。     

叶绿体转化及其用于疫苗表达研究的最新进展

随着植物转基因研究的不断深入,核基因组转化的转基因沉默现象严重影响了基因工程的应用效果.植物叶绿体遗传转化以叶绿体基因组为平台对植物进行遗传操作,外源基因定点整合及母性遗传特性能较好地解决＂顺式失活＂和＂位置效应＂等类的基因沉默问题和转基因逃逸等安全问题,成为植物基因工程发展的新方向,在工业、农业及医药生物领域发挥了重要作用,也为生产廉价、安全的植物疫苗提供了新思路.本文在简要介绍叶绿体转化的原理、转化方法与优势的基础上,重点综述了近年来通过该技术表达的一些重要的病毒抗原和细菌抗原.最后,对叶绿体转化技术在表达外源基因方面存在的问题进行分析.未来随着叶绿体基因表达、调控机制研究的逐渐深入及相关技术体系的日臻完善,叶绿体转化有望成为疫苗生产的生力军.     

11种植物psbA基因的密码子偏好性及聚类分析

植物叶绿体psbA基因的启动子是叶绿体基因工程中常用的启动子,研究该基因的编码特点对完善叶绿体基因工程的研究设计、提高外源基因在受体物种中高效、稳定的表达具有重要作用。本研究综合运用了多种分析软件,对11种植物的叶绿体psbA基因进行了分析。结果表明,11种植物psbA基因的ENC（Effective Number o...     

黄瓜、甜瓜和西瓜MLO基因家族的比较基因组学分析

MLO基因是植物特有的一类基因家族,在调控植物抵抗生物和非生物胁迫方面起着重要作用。为了揭示葫芦科作物MLO基因的遗传变异及系统发育关系,本文以黄瓜、甜瓜和西瓜基因组数据为基础,利用生物信息学方法对其MLO基因家族进行鉴定与分析。结果发现,黄瓜、甜瓜和西瓜基因组中共含有42个MLO基因家族成员,每一个物种均含有14个成员,且保守性强;系统发育关系揭示了这些成员在黄瓜、甜瓜和西瓜基因组中并不呈现一一对应关系,表明MLO型基因在这3种植物分化之后分别发生了扩展和丢失;进一步将其与模式植物拟南芥、番茄、豌豆MLO型白粉病基因进行聚类分析:一方面,借助于拟南芥MLO基因的分类标准,揭示了在黄瓜、甜瓜和西瓜基因组中也存在双子叶植物MLO型白粉病基因的特异区组,他们各自至少包含3个候选的白粉病基因,序列比对进一步发现这些基因均具有MLO型白粉病基因的典型结构特征,如7个跨膜结构域、钙调蛋白结合区以及两个缩氨酸区域(I和II);另一方面,大部分区组中包含的MLO基因均来源于拟南芥和黄瓜、甜瓜和西瓜MLO基因家族的成员,表明了MLO基因家族在拟南芥和葫芦科作物分化之前就已经存在。EST表达分析表明MLO基因广泛地参与到黄瓜、甜瓜和西瓜器官的营养生长和生殖生长。研究结果为揭示黄瓜、甜瓜和西瓜MLO基因的进化关系、功能及克隆表达分析奠定了基础。     

基于图像配准分析物种进化关系的新方法

图像配准是图像处理的一个重要技术,可用于分析两幅图像之间的相似度.本文提出了一种基于图像配准分析物种进化关系的新方法：首先利用一阶马尔可夫链方法计算不同基因组序列的寡聚核苷酸转移概率矩阵;然后将转移概率矩阵转换为彩色图像矩阵,并绘制物种两两之间彩色图像矩阵的联合直方图;最后分析联合直方图点集的分布情况,引入直方图点集的散度公式,将其作为相似性测度的标准,从而鉴定物种亲缘关系的远近.100种细菌全基因组的计算结果表明,相较于单基因法或基于基因组寡聚核苷酸频率组分差异信息的方法,本文提出的新方法具有更高的准确度和分辨力,它不仅能够很好地分辨科以下的分类单元,而且对科以上的分类单元同样具有较好的区分效果.该方法有望发展成为物种鉴定及系统发育推断的有效手段.     

GFP叶绿体转化烟草茎叶降解对土壤微生物数量及土壤酶活性的影响

以GFP叶绿体转基因烟草及对照烟草（非转基因烟草）为材料,在实验室条件下研究了叶绿体转基因烟草及对照烟草（非转基因烟草）在茎叶降解过程中及完全降解后对土壤微生物主要类群（细菌、真菌、放线菌）的影响,并对相关的土壤酶活性进行了分析。结果表明,在烟草茎叶降解过程中及完全降解后：（1）叶绿体转基因烟草及对照烟草（非转基因烟草）对微生物的数量影响不显著;（2）土壤微生物总量相比为细菌〉放线菌〉真菌;（3）叶绿体转基因烟草茎叶降解对土壤酶活性没有显著影响;（4）GFP基因没有水平转移到土壤微生物基因组中。以上结果显示GFP叶绿体转化烟草茎叶降解对土壤微生物数量及土壤酶活性没有显著的影响。     

豇豆叶绿体基因组密码子使用偏好性分析

为了探究豇豆（Vigna unguiculata）叶绿体基因组密码子的使用模式,本研究从NCBI下载完整的豇豆叶绿体基因组序列,并对其进行结构分析。利用CodonW和CUSP对筛选获得的50条可编码蛋白序列（CDS）进行分析,获得GC1、GC2、GC3、RSCU、CAI、CBI、Fop、ENc、RFSC等重要参数,并进行中性绘图、PR2-plot绘图、ENc-plot绘图以及对应分析、最优密码子分析和其他物种对比分析。结果发现,豇豆叶绿体基因密码子更偏向以A或U(T)结尾,G和C在密码子各位置中的占比较低,平均值为36.31%;有效密码子数（ENc）的平均值为44.903,密码子偏好性较弱;GC1与GC2、GC3间均有相关性,表明碱基突变对密码子选择也有影响。从中性绘图、PR2-plot绘图、ENc-plot绘图结果可以看出,豇豆叶绿体密码子使用偏好性同时受到了碱基突变与自然选择的影响。本研究最终筛选出20个最优密码子,并将豇豆叶绿体基因组密码子使用频率与其他物种进行比较,发现豇豆与番茄在密码子使用频率上存在较高的相似度。本研究结果为提高豇豆叶绿体外源基因的表达效率提供了参考依据。     

不同种源马蔺的叶绿体DNA变异研究

本研究利用通用引物,采用PCR技术从9个不同地理种源的马蔺样品中扩增了叶绿体基因组trnLt-rnF间隔区的DNA片段,进行了序列测定,并运用DNAMAN软件分析了不同种源马蔺的亲缘关系。结果表明:9个马蔺植物样品分为2个相对独立的组,其中第1组包括采自北京、泰山、武威、涿洲、民勤、固原6个种源的马蔺,第2组包括采自太仆、乌鲁木齐和西乌旗3个种源的马蔺。2个组的亲缘关系很远,同源性只有45%,这表明马蔺物种不是单系起源,本研究结果也表明了利用叶绿体基因间序列研究马蔺亲缘关系是可行的。     

PHAs合酶phaC2基因转化烟草叶绿体的研究

中长链羟基脂肪酸聚酯 (medium-chain-length-PHAs, mcl-PHAs) 属于微生物聚酯。Ⅱ型PHA合酶是mcl-PHAs生物合成途径中的关键酶。将编码Ⅱ型PHA合酶的基因phaC2与水稻(Oryza sativa )叶绿体psbA基因的启动子和终止子构建表达盒(RpsbA-pro-phaC2-RpsbA-ter)，连同壮观霉素抗性基因aadA表达盒(prrn-aadA-TpsbA-ter)一起克隆进烟草叶绿体基因组同源片段rbcL和accD中，构建了烟草叶绿体转化表达载体pTC2。基因枪法转化烟草(Nicotiana tobacum L.)叶片，获得具有壮观霉素抗性的转基因烟草。对T0代和T1代转基因植株的PCR和Southern blot鉴定结果表明, 外源基因确已整合进烟草叶绿体基因组中，T1代转基因植株已达同质化。RT-PCR分析结果证实phaC2基因已在转录水平上表达。转基因植株的正反交试验证明外源基因在转基因后代中遵循母性遗传规律，不存在转基因的花粉漂移现象。通过叶绿体遗传转化, 实现中长链羟基脂肪酸聚酯合成关键酶基因phaC2在烟草叶绿体基因组中的稳定转化。     

Cytoplasmic diversity of the cotton genus as revealed by chloroplast microsatellite markers

The diversity of chloroplast genomes has played an important role, as have those of nuclear and mitochondrial genomes, in the evolution of plants. The sequences of the chloroplast genome supply unsubstituted information for genome analysis. In order to understand the genetic differentiation and relationship of cotton species, we investigated the cytoplasmic diversity of chloroplast genomes in 41 Gossypium accessions with 75 chloroplast simple sequence repeat (cpSSR) markers. The markers were developed from reference sequences of the chloroplast genomes of G. hirsutum and G. barbadense and covered approximately 12.6 kb. Among the 75 markers, 50 were polymorphic, with polymorphism information content values ranging from 0.11 to 0.88. Analyses of the dataset demonstrated that single copy regions were much more informative than inverted repeat regions. The non-coding sequences were well differentiated among these species. For some common cpDNA haplotypes, the E-genome species that may be the oldest of the extant cotton species was deduced. The differentiation of A-genome species lagged behind that of AD-genome species. Neither G. herbaceum nor G. arboreum was the cytoplasmic donor of tetraploid species, strongly suggesting that AD genomes originated from an extinct ancestor of modern A-genome species. We speculate that the genetic differentiation of the chloroplast genome of each cotton species resulted from the dispersal of that species and its adaptations to local ecological conditions. These cpSSR markers provided valuable information to reveal the diversity and differentiation of cotton during evolution.     

Characterizing the cytoplasmic diversity and phyletic relationship of Chinese landraces of soybean, Glycine max, based on RFLPs of chloroplast and mitochondrial DNA

Restriction fragment-length polymorphism (RFLP) of chloroplast and mitochondrial DNA of many soybean landraces were analyzed to characterize their cytoplasmic genome and to consider the diversity and phylogeny of soybean in China. Three chloroplast and five mitochondrial types were distinguished, and seven cytoplasmic genome types were observed in different combinations in specimens collected from China. The cytoplasmic genome diversity was higher at the Yangtze River Valley. The main group of cytoplasmic genomes was composed of five types related phylogenetically and occurred in the southern region of China. Two unique types were very different in cytoplasmic genome constitution from each other and from the main group. They were considered to be derived from wild plants with the same cytoplasmic genome and to occur in two remote regions, the northern region and the Yangtze River Valley of China, respectively. Therefore, when considered with regard to the cytoplasmic genome of soybean landraces detected in China, seven genome types are distinguished, some types may be assumed to have arisen in wild soybean before domestication; and the Yangtze River Valley is the center of cytoplasmic diversity of the soybean.     

Mitochondrial DNA RFLP in genus Oryza and cultivated rice

Summary Ninety-three accessions representing 23 species from the genus Oryza were surveyed for restriction fragment length polymorphism (RFLP) in mitochondrial (mt) DNA by probing total DNA with 15 known mt sequences cloned in plasmids from higher plants, and five mt genomic cosmid clones from maize. Very low levels of intra-specific and even intra-cytologically-defined nuclear genome mt DNA RFLP were found. High between-genome differentiation appeared, suggesting phylogenetic relationships consistent with data from previous nuclear and chloroplast (cp) DNA studies. Parallel inheritance of cp and mt DNA was found. There was one major exception: the mt DNA of the allotetraploid CD genome is apparently equally related to two putative diploid progenitors, which is suggestive of an interspecific recombination.RRLP in mt DNA was also probed in 82 cultivars, with four plasmid probes. Some bands not seen in the wild species appeared in O. sativa, with intra-specific polymorphism relatively higher than in the wild species. The pattern of variation paralleled that at the cp DNA level between the indica and japonica subspecies.     

Micromonospora: An important microbe for biomedicine and potentially for biocontrol and biofuels

Micromonospora species have long been recognized as important sources of antibiotics and also for their unusual spores. However, their involvement in plant-microbe associations is poorly understood although several studies demonstrate that Micromonospora species function in biocontrol, plant growth promotion, root ecology, and in the breakdown of plant cell wall material. Our knowledge of this generally understudied group of actinomycetes has been greatly advanced by the increasing number of reports of their associations with plants, by the deployment of DNA cloning and molecular systematics techniques, and by the recent application of whole genome sequencing. Efforts to annotate the genomes of several Micromonospora species are underway. This information will greatly augment our knowledge of these versatile microorganisms.     

Using diversity of the chloroplast genome to examine evolutionary history of wheat species

Chloroplast microsatellites (SSRs) are conserved within wheat species, yet are sufficiently polymorphic between and within species to be useful for evolutionary studies. This study describes the relationships among a very large set of accessions of Triticum urartu Thum. ex Gandil., T. dicoccoides (Körn. ex Asch. et Graebn.) Schweinf., T. dicoccon Schrank, T. durum Desf., T. spelta L., and T. aestivum L. s. str. based on their cpSSR genotypes. By characterising the chloroplast diversity in each wheat species in the evolutionary series, the impact on diversity of major evolutionary events such as domestication and polyploidyisation was assessed. We detected bottlenecks associated with domestication, polyploidisation and selection, yet these constrictions were partially offset by mutations in the chloroplast SSR loci that generated new alleles. The discrete cpSSR alleles and haplotypes observed in T. urartu and Aegilops tauschii, combined with other species specific polymorphisms, provide very strong evidence that concur with current opinion that neither species was the maternal and thus cytoplasmic donor for polyploid wheats. Synthetic hexaploid wheats possessed the same chloroplast haplotypes as their tetraploid progenitors demonstrating how the novel synthetic wheat lines have captured chloroplast diversity from the maternal parents, the chloroplast is maternally inherited and novel alleles are not created by genomic rearrangements triggered by the polyploidisation event.     

油菜叶绿体多顺反子定点整合表达载体的构建

根据拟南芥叶绿体基因组序列设计PCR引物,从我国甘蓝型油菜栽培品种F4叶绿体基因组中克隆了长度为1.5kb的trnI和trnA2个基因序列,核酸序列分析表明它们与拟南芥基因的同源性高达94%和99%,这两个克隆的油菜叶绿体基因组序列trnI和trnA被用于构建叶绿体定点整合表达载体。利用烟草叶绿体基因强启动子Prrn和终止子TpsbA,以及筛选标记基因aadA和目的基因HSA,构建了多顺反子表达盒Prrn-aadA-HSA-TpsbA,将表达盒置于油菜叶绿体trnI和trnA序列之间,最后构建成油菜叶绿体多顺反子定点整合表达载体pIPaHTA。酶切鉴定及序列分析证实,构建的表达载体具有预期的调控元件及结构,这为后期油菜叶绿体转化体系的建立奠定了基础。     

Identification of <Emphasis Type="Italic">Aegilops</Emphasis> L. species and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. based on chloroplast DNA

The genus Aegilops L. is a very important genetic resource for the breeding of bread wheat Triticum aestivum. Therefore, an accurate and easy identification of Aegilops species is required. Traditionally, identification of Aegilops species has relied heavily on morphological characters. These characters, however, are either not variable enough among Aegilops species or too plastic to be used for identification at the species level. Molecular markers that are more stable within species, therefore, could be the alternative strategy towards an accurate identification. Since the chloroplast DNA has a lower level of evolution compared to the nuclear genome, an attempt was made in this study to investigate polymorphism in the chloroplast DNA among 21 Aegilops species (including Ae. mutica that is now known as Amblyopyrum muticum) and between the latter and T. aestivum to generate markers for the diagnosis of all targeted species. Cleaved amplified polymorphic sequence (CAPS) applied on 22 coding and non-coding chloroplast regions using 80 endonucleases and sequencing of two of those regions revealed little polymorphism between T. aestivum and the various Aegilops species examined and to a less extent was the variation among Aegilops species. Polymorphism observed among species analysed allowed the discrimination of T. aestivum and 12 Aegilops species.     

Polymorphisms Among Chloroplast and Mitochondrial Genomes of <Emphasis Type="Italic">Citrullus</Emphasis> Species and Subspecies

Twelve and six DNA clones representing various parts of chloroplast and mitochondrial genomes, respectively, were used to detect polymorphism among five watermelon cultivars and 21 U.S. Plant Introductions (PIs) collected from diverse geographical locations and representing major groups of Citrullus species. Cluster analysis based on 20 chloroplast DNA (cpDNA) and 10 mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) markers differentiated the accessions into three major phenetic groups: PIs and watermelon cultivars of Citrullus lanatus subsp. vulgaris (Schrad. ex Eckl. et Zeyh.) Fursa (also designated as C. lanatus var. lanatus) (group I), PIs of C. lanatus var. citroides (of C. lanatus subsp. lanatus Schrad. ex Eckl. et Zeyh.)(group II), and C. colocynthis (L.) Schrad. PIs (group III). The chloroplast and mitochondrial genomes of watermelon cultivars are distinct, but closely related to those of the C. lanatus var. lanatus PIs. On the other hand, the chloroplast and mitochondrial genomes of the wild species C. colocynthis are more similar to those of C. lanatus var. citroides. Polymorphic cpDNA and mtDNA markers identified in this study can complement isozyme and nuclear DNA data used in earlier phylogenetic and phenetic classifications of Citrullus PIs. These cpDNA and mtDNA markers are being used in experiments designed to enhance watermelon cultivars by replacing the chloroplast and mitochondrial genome of cultivated watermelon with those of the wild species C. colocynthis.