基于叶绿体16S rRNA的绿色裸藻类系统发育及性状进化研究

该研究基于叶绿体16S rRNA基因序列,构建绿色裸藻类的系统发育树,并对绿色裸藻类植物8个形态性状进行祖先重建分析,以明确绿色裸藻类植物的系统演化关系,为研究该类植物的起源提供理论依据。结果表明：(1)贝叶斯法构建的绿色裸藻类系统发育树显示,双鞭藻属与拟双鞭藻属互为姐妹群,扁裸藻属、鳞孔藻属和盘裸藻属亲缘关系较近,而囊裸藻属和陀螺藻属亲缘关系较近,裸藻属、隐裸藻属、柄裸藻属和旋形藻属亲缘关系较近,表明裸藻属不是一个单系类群。(2)基于形态性状的祖先重建结果显示,绿色裸藻类相对原始的7个性状包括：表质柔软易变形,出现螺旋形线纹,细胞后端渐尖或尖尾刺状,无囊壳,叶绿体为片状、盾状或大盘状,具无鞘蛋白核,副淀粉粒为小颗粒状且数量不定,而鞭毛长度不能推断可能的祖先状态。(3)综合8种性状祖先重建结果发现,裸藻属和眼裸藻属植物具有所有原始性状,可能是最先出现的绿色裸藻类的祖先。     

两种裸藻的无叶绿体突变株

利用Ofloxacin处理获得了纤细裸藻和中型裸藻无叶绿体突变株。吸收光谱测定和自荧光显微观察证明突变株无叶绿素合成,DAPI染色方法显示突变株细胞不存在质体DNA,而无色的长变胞藻细胞中仍有可检测质体DNA,说明二突变株质体已消失。SDS－PAGE显示野生种和突变株各有特异蛋白表达。     

绿眼虫的叶绿体

绿眼虫 (Euglena viridis Ehrenberg1830 )为 1种习见鞭毛虫 ,植物学中裸藻门裸藻属的叶绿体特征为“细胞内有许多颗粒状叶绿体 ,分布于原生质近表面 ,称边缘位叶绿体 ,少数种类为中轴位的星状叶绿体 ,数目很少 ,只有 1～ 2个”。国内许多版本 (195 0～ 1992 )《动物学》、《普通动物学》、《无脊椎动物学》、《大学动物学》等对绿眼虫的叶绿本形态描述多为 :“大量圆形叶绿体”、“多个叶绿体”、“有甚多含叶绿素的色素体”,“很多色素体 ,含多量叶绿素”等。陈义《动物学》(195 0 )中明确提出“绿眼虫的色素体 1个 ,在体之中央 ,胞核之…     

中国眼裸藻属(Euglenaria)分类学研究

为对眼裸藻属Euglenaria Karnkowska, Linton&Kwiatowski进行分类学修订,研究以采自中国的12株3种眼裸藻属物种为材料,基于形态学和分子生物学手段对眼裸藻属进行系统发育研究,鉴定了3种眼裸藻属物种,分别是尾眼裸藻Euglenaria caudata (Hubner) Karnkowska et Linton、项圈眼裸藻Euglenaria anabaena(Mainx) Karnkowska et Linton和棒形眼裸藻Euglenaria clavata (Skuja) Karnkowska et Linton,提供了它们的形态特征及分子数据。基于最大似然法和贝叶斯法构建了系统发育树,以亲缘关系较近的双鞭藻科Eutreptiaceae Hollande物种为外类群,其余的裸藻类分为扁裸藻科Phacaceae Kim, Triemer&Shin和裸藻科Euglenaceae Dujardin物种两大支,眼裸藻属位于裸藻科分支(0.84/-),共包含28株眼裸藻属物种,主要分为3个小分支,每个小支的支持率均高达99%以上,项圈眼裸藻和尾...     

衣藻叶绿体遗传转化技术的研究

单细胞真核生物衣藻（Chlamydomonas）只有一个大型的杯状叶绿体,占细胞总体积的40％．叶绿体基因组全长196Kb,含有两个21Kb的重复序列．由于其结构简单,易于培养,遗传背景清楚,因而它一直是研究先合作用和叶绿体遗传学的模式植物。本文主要就在藻叶绿体遗传转化的方法和技术、筛选标记及应用等方面的研究进展作一综述,1衣菜叶绿体转化中的外源DNA导入技术1．1基因枪法1987年,Sanford等发明了高速徽次轰击法这一新的DNA导人技术。其方法是：火药或氦气驱动承载有微细金粉或鹤担的自料股,随后它担击在一张阻挡它的金属同一一,高…     

藻类的前世今生——浅析内共生学说与藻类的进化

正什么是藻类?藻类是一类有趣的低等生物的统称。它们没有根茎叶分化,绝大多数能够进行光合作用。说它们低等,是因为在它们完整的生命周期内自始至终不会形成胚,而高等生物(例如种子植物和哺乳动物等)则会在繁殖发育过程中形成胚。藻类既包含能进行光合作用的细菌——蓝藻(蓝细菌),也包括能够进行光合作用的低等真核生物(灰胞藻、绿藻、红藻、隐藻、定鞭藻、异鞭藻、甲藻、裸藻和网绿藻等),还包括具有叶绿体但不能进行光合作用的     

杜氏盐藻完整叶绿体的分离及其蛋白提取

应用高压破碎及蔗糖密度梯度离心的方法,分离出杜氏盐藻的完整叶绿体,随后用冻融法和研磨法分别提取叶绿体蛋白,并通过蛋白定量和SDS-PAGE凝胶电泳对两种蛋白提取方法进行比较,确立了一套适用于杜氏盐藻叶绿体分离和蛋白提取、定量以及电泳的方法.结果表明:用高压破碎结合蔗糖密度梯度离心的方法能够获得完整且较纯的盐藻细胞叶绿体;SDS-PAGE凝胶电泳结果显示,冻融法提取叶绿体蛋白效率高,电泳条带清晰,蛋白数量较多,蛋白齐全.为下一步在亚细胞水平进行杜氏盐藻耐盐机制的蛋白组学研究奠定了基础.     

珠三角河网裸藻的多样性特征

珠三角河网是珠江汇入南海的必经之地,2012年对该水域裸藻门及各属的多样性特征及影响因子进行系统阐析。调查期间共发现裸藻12属84种,其中裸藻属是最主要类群,其次为扁裸藻属和囊裸藻属。珠三角河网独特的地理位置和人类活动造成的水体富营养化是该水域裸藻物种多样性丰富的主要原因。季节特征显示,丰水期裸藻的种类丰富度和生物量均明显高于枯水期。除了洪水引发的裸藻物种的外源供给增大外,泄洪所带来的有机质的增加也是有利因素。此外,丰水期对应的高温也是有益条件。空间特征存在明显的季节差异,丰水期河网外侧站位的生物量和种类丰富度高于中部站位,主要原因是受径流影响较大;枯水季节除广州附近站位的值明显偏高外,其它站位间差异不大,营养盐的空间格局是主要决定因素。尽管裸藻主要依赖于沿江静水水体中的外源供给,分析表明,其在随水流下行的过程中存在增长过程。不同藻属相对组成的结果显示,扁裸藻属和囊裸藻属因属于生长缓慢的K-选择策略型,富集营养物质的能力不及裸藻属,仅在枯水期与裸藻属形成互补优势。裸藻门及主要属的生物量与种类丰富度呈线性正相关关系,偏重于多样性单峰模式的上升区。     

高浓度钾对杜氏盐藻叶绿体超微结构的影响

在观察到高浓度K 对杜氏盐藻 (DunaliellasalinaTeod .)生长有显著抑制现象 ,以及初步证明高浓度钾通过抑制杜氏盐藻的光合作用而抑制其生长的基础上 ,探讨了高浓度K 对杜氏盐藻叶绿体超微结构的可能影响。电镜观察表明 ,培养液中加入 10 0mmol/LKCl使叶绿体膨胀 ,类囊体膨大并解体 ,叶绿体中同时大量形成及积累淀粉粒。高pH对盐藻叶绿体超微结构有类似影响。高钾引起的叶绿体超微结构的改变及叶绿体中淀粉粒的积累可能是高K 抑制盐藻光合作用及生长的原因之一。     

温度和食用藻密度对发头裸腹潘种群动态和两性生殖的影响

研究了不同温度和食用藻密度对发头裸腹潘种群动态和两性生殖的影响.结果表明:温度、食用藻密度对发头裸腹溞的种群密度、雄体密度和卵鞍数均有显著影响.高食用藻密度组的发头裸腹潘种群密度明显高于中、低食用藻密度组,其最大种群密度出现在20℃下的高食用藻密度组.在相同的温度下,发头裸腹溞的首次产幼溞数随食用藻密度的降低而减少,平均每个母潘首次产出的最大幼潘数出现在25℃下的高食用藻密度组.高食用藻密度组发头裸腹潘产生的雄体密度明显高于中、低食用藻密度组.发头裸腹溞的雄体密度与其种群密度之间存在极显著的相关性.发头裸腹溞所产的卵鞍数随食用藻密度的下降而下降,且25℃下发头裸腹溞所产的卵鞍数明显高于其他温度组.与温度相比,食用藻密度对发头裸腹潘的种群动态和两性生殖的影响更大.     

高等植物叶绿体表达重组蛋白研究进展

近年来,基因工程技术发展迅速,许多重组蛋白得以表达。其中利用植物生物反应器表达特异药物蛋白为人类一些重要疾病的预防和治疗提供了新途径。植物叶绿体遗传转化和表达系统成为目前植物生物反应器的研究热点。因结构和遗传上的特殊性,高等植物叶绿体在重组蛋白表达方面具有独特优势,外源基因表达量高、定点整合,而且叶绿体母系遗传特性保证了生物安全性。很多重要药用蛋白质在植物叶绿体中表达成功。烟草作为高等植物叶绿体转化模式植物,在疫苗抗原、抗体等药物蛋白和其他重要重组蛋白表达方面取得显著进展。高等植物叶绿体遗传转化也为叶绿体基因的表达和调控机制的研究提供新的技术和方法。文中从叶绿体遗传转化原理、载体构建、重组蛋白和重要药物蛋白在叶绿体中的表达以及重组蛋白表达对植物代谢和性状影响等多个角度,对高等植物叶绿体遗传转化体系研究的新进展进行了综述,以期为叶绿体表达平台的开发和重要药用蛋白质的表达提供新思路。     

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。     

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, polarity, cloning, and evidence for an intervening sequence

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.     

Genetic engineering of algal chloroplasts: Progress and prospects

The last few years has seen an ever-increasing interest in the exploitation of microalgae as recombinant platforms for the synthesis of novel bioproducts. These could be biofuel molecules, speciality enzymes, nutraceuticals, or therapeutic proteins, such as antibodies, hormones, and vaccines. This exploitation requires the development of new genetic engineering technologies for those fast-growing, robust species suited for intensive commercial cultivation in bioreactor systems. In particular, there is a need for routine methods for the genetic manipulation of the chloroplast genome, for two reasons: firstly, the chloroplast genetic system is well-suited to the targeted insertion into the genome and high-level expression of foreign genes; secondly, the organelle is the site of numerous biosynthetic pathways and therefore represents the obvious “chassis,” on which to bolt new metabolic pathways that divert the carbon fixed by photosynthesis into novel hydrocarbons, pigments, etc. Stable transformation of the algal chloroplast was first demonstrated in 1988, using the model chlorophyte, Chlamydomonas reinhardtii. Since that time, tremendous advances have been made in the development of sophisticated tools for engineering this particular species, and efforts to transfer this technology to other commercially attractive species are starting to bear fruit. In this article, we review the current field of algal chloroplast transgenics and consider the prospects for the future.     

Restriction endonuclease map of Euglena gracilis chloroplast DNA.

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.     

Isolation of Euglena gracilis chloroplast 5S ribosomal RNA and mapping the 5S rRNA gene on chloroplast DNA.

Ribosomal RNA (5S) from Euglena gracilis chloroplasts was isolated by preparative electrophoresis, labeled in vitro with 125I, and hybridized to restriction nuclease fragments from chloroplast DNA or cloned chloroplast DNA segments. Euglena chloroplast 5S rRNA is encoded in the chloroplast genome. The coding region of 5S rRNA has been positioned within the 5.6 kilobase pair (kbp) repeat which also codes for 16S and 23S rRNA. There are three 5S rRNA genes on the 130-kbp genome. The order of RNAs within a single repeat is 16S-23S-5S. The organization and size of the Euglena chloroplast ribosomal repeat is very similar to the ribosomal RNA operons of Escherichia coli.     

Evidence for the nuclear location of the gene for chloroplast elongation factor G.

The chloroplast protein synthesis factor responsible for the translocation step of polypeptide synthesis on chloroplast ribosomes (chloroplast elongation factor G [EF-G]) has been detected in whole cell extracts and in isolated chloroplasts from Euglena gracilis. This factor can be detected by its ability to catalyze translocation on 70 S prokaryotic ribosomes such as those from E. coli. Chloroplast EF-G is present in low levels when Euglena is grown in the dark and can be induced more than 20-fold when the organism is grown in the light. The induction of this factor by light is inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. However, inhibitors of chloroplast protein synthesis such as streptomycin or spectinomycin have no effect on the induction of this factor by light. Furthermore, chloroplast EF-G can be partially induced by light in an aplastidic mutant (strain W₃BUL) which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-G resides in the nuclear genome, and that this protein is synthesized on cytoplasmic ribosomes prior to compartmentalization within the chloroplasts.     

叶绿体基因组研究进展

作为植物细胞器的重要组成部分和光合作用的器官,叶绿体在生物进化的漫长历史中发挥了重要作用.伴随着生物技术的深入发展,人们发现叶绿体基因组结构和序列的信息在揭示物种起源、进化演变及其不同物种之间的亲缘关系等方面具有重要价值.与此同时,比核转化具有明显优势的叶绿体转化技术在遗传改良、生物制剂的生产等方面显示出巨大潜力,而叶绿体基因组结构和序列分析则是叶绿体转化的基石.基于叶绿体的这些重要作用,收集整理了有关的资料,从几个方面归纳了本领域最近的研究进展,希望能使读者对迅速发展的叶绿体基因组研究有更全面的了解,以及对叶绿体基因组在物种的进化、遗传、系统发育关系等方面的作用有更深刻的认识,同时也希望对叶绿体转化技术的研究和广泛应用产生积极作用.     

Transgenic plastids in basic research and plant biotechnology

Facile methods of genetic transformation are of outstanding importance for both basic and applied research. For many years, transgenic technologies for plants were restricted to manipulations of the nuclear genome. More recently, a second genome of the plant cell has become amenable to genetic engineering: the prokaryotically organized circular genome of the chloroplast. The possibility to directly manipulate chloroplast genome-encoded information has paved the way to detailed in vivo studies of virtually all aspects of plastid gene expression. Moreover, plastid transformation technologies have been intensely used in functional genomics by performing gene knockouts and site-directed mutageneses of plastid genes. These studies have contributed greatly to our understanding of the physiology and biochemistry of biogenergetic processes inside the plastid compartment. Plastid transformation technologies have also stirred considerable excitement among plant biotechnologists, since transgene expression from the plastid genome offers a number of most attractive advantages, including high-level foreign protein expression and transgene containment due to lack of pollen transmission. This review describes the generation of plants with transgenic plastids, summarizes our current understanding of the transformation process and highlights selected applications of transplastomic technologies in basic and applied research.