【作者】 崔翠菊；

【导师】 何光源； 杨广笑；

【作者基本信息】 华中科技大学 ， 生化与分子生物学， 2010， 博士

【摘要】 植物细胞中,除了细胞核中具有遗传物质DNA之外,叶绿体和线粒体也拥有半自主性的能自我复制和遗传的基因组。随着基因组测序技术的发展,越来越多的植物核基因组、叶绿体基因组和线粒体基因组相继完成了测序。研究发现叶绿体基因组和线粒体基因组结构和序列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,使得叶绿体基因工程成为植物基因工程中发展的新领域。叶绿体基因组结构和序列分析则是叶绿体基因工程的基础,而叶绿体基因工程研究又为细胞器与核基因组之间的基因转移、基因表达的协调作用机制等研究提供实践证明,对研究植物的进化起源有重要的意义。叶绿体基因工程技术也随着叶绿体测序的完成逐渐成为转基因定点整合、多拷贝高效表达外源基因,培育优良性状农作物品种和作为生物反应器合成目的蛋白的热门选择。本研究以小麦叶绿体基因组中的atpB和rbcL作为同源重组片段,用nptII和gfp分别作为筛选基因和标记基因,构建小麦叶绿体定点整合表达载体,用基因枪轰击法分别转化小麦的幼胚盾片和幼穗段。对T0代再生植株进行分子生物学验证,PCR和Southern blot结果证实标记基因gfp已经成功整合到叶绿体基因组中,并得到了同质化的再生植株。用T1代植株的叶片检测到GFP绿色荧光在叶绿体中表达,证明已经成功建立了小麦叶绿体稳定遗传转化体系。应用植物细胞作为生物反应器生产药用蛋白或疫苗等也成为现今研究的热点,本研究用农杆菌浸染烟草叶片的方法将人干扰素α-2b基因转入烟草核基因组,在再生烟草植株的叶片蛋白中检测到具有活性的人干扰素α-2b蛋白;构建了烟草叶绿体定点整合载体,为以烟草叶绿体表达人干扰素α-2b蛋白做好了前期准备。本文全面统计分析研究了已完成测序的藻类,苔藓,蕨类,裸子,被子植物的各物种的核基因组和细胞器基因组的相关信息。分别比较了不同物种的基因组大小,基因编码区和非编码区的大小变化趋势。在174种叶绿体基因组已测序的物种中,有26种核基因组也完成了测序,分别比对各物种的叶绿体基因组和它的核基因组,结果发现随着物种越来越高等,其叶绿体基因在核基因组中的同源性也越来越高,有5种被子植物的同源性高达100%。对15种线粒体基因组和核基因组都已测序的物种进行同样的全序列比对,也发现了随物种进化逐渐增高的趋势,但与叶绿体相比,不是太明显,最高的同源比例仅为82.6%。本文还对藻类、双子叶植物和单子叶植物的叶绿体的保守基因进行了统计。将单子叶植物保守基因中没有的藻类的保守基因分别与拟南芥和水稻的核基因组比对分析,其中绝大多数遗传系统基因都成功转移,而光合系统和生物合成系统则都丢失了相当一部分基因。还对拟南芥和水稻的叶绿体基因和线粒体基因在核基因组中的同源比例和拷贝数进行了统计。综合分析这些序列的信息后,我们提出以下假设,在植物进化过程中,细胞器基因到核基因组的转移,大致经过了先以拷贝的形式转移到核基因组的非编码区,而后获得表达功能,细胞器中相应基因的消失进而使细胞中的遗传物质逐步转移到核基因组中,最终实现核基因组对植物遗传物质表达的总体调控功能。更多还原

【Abstract】 In plant cells, in addition to the nucleus with the genetic material DNA, two kinds of organelles, chloroplast and mitochondria also have a semi-autonomous self-replicable and heritable genome. With the advances in DNA sequencing technology, more and more plant nuclear genome, chloroplast genome and mitochondrial genome sequencing projects have been completed. Previous studies have shown that the genome structure and gene information in chloroplasts and mitochondria could provide important reference on the origin of plant species, evolution and the genetic relationship among different species. Meanwhile, the chloropolast genetic engineering has become a novel developmental field of plant genetic engineering as its advantages over nuclear transformation in the area of genetic improvement of crops and expression of therapeutic proteins. The structure and sequences analysis of chloroplast genome is the foundation of chloroplast genetic engineering. To some extent, this kind of research can not only provide experimental evidence for the mechanism of coordination gene expression between nuclear and organellar genomes, but helpful for understanding of the plant species origin and gene transfer events in the evolution process as well.Chloroplast genetic engineering technology offers a novel method to carry out sit-specific integration, highly effective gene expression, breeding for various resistant crops and creating bioreactor for biosynthesis of certain valuble proteins. In this study, two wheat chloroplast genes, atpB and rbcL, were chosen as homologous recombination fragment, with the nptII and gfp gene as selection and marker genes to construct site-specific integration vector for wheat chloroplast transformation. The fertile, homoloplasmic regeneration wheat plants were obtained by bombarding the immature inflorescences and scutella from immature embryos. PCR and Southern blot analysis of T0 plants demonstrated that gfp gene has successfully been integrated into the wheat chloroplast genome. And the expression of green fluorescent protein (GFP) was confirmed by confocal microscopy in the chloroplast of the T1 seedlings. The above results demonstrated that a stable genetic transformation system of wheat chloroplasts has been established.Using plant cell as a bioreactor to produce pharmaceutical proteins or vaccines, also has emerged as a hot research. In this research part, human interferonα- 2b (HuIFNα-2b) gene was transformed into the nuclear genome of tobacco leaves through the leaf disc dip method and the molecular analysis confirmed that the regenerated tobacco plants could express the active HuIFNα-2b protein. Tobacco chloroplast transformation vector containing the HuIFNα-2b gene was also successfully constracted and it would be applied to potentially express the HuIFNα-2b protein in tobacco chloroplasts.In this study, comprehensive statistics of nuclear and organelles genome information has been carried out among the sequenced species of Algae, Bryophytes, Pteridophytes, Gymnosperm and Angiosperm. The variety trend of the size of genome, gene-coding region and non-coding region during their evolutionary course was compared. In 174 species of which, their chloroplast genome sequenced, 26 species of nuclear genome sequencing were also completed. Aligning the various species of chloroplast genome and its nuclear genome and it was found that the higher of the species, the higher homology between the chloroplast DNA and its nuclear genome are. There are five species of Gymnosperm group whose homology were up to 100%. The similar analysis of 15 species of both sequenced mitochondrial DNA and nuclear genome showed the same trend with the chloroplast genome alignment. But the increasing extent in mitochondrion was not so obvious and the highest proportion was only 82.6%.The conserved genes in chloroplast genomes of algae, dicotyledons and monocotyledons species were statistically counted. The conserved genes in Algae but not present in Monocotyledons were compared with the nuclear genomes of Arabidopsis thaliana and Oryza sativa respectively, the majority genes of genetic system were successfully transferred, and considerable genes of the biosynthesis and photosynthetic system have lost. Statistics of the proportion and copy number of chloroplast and mitochondrial genes in their nuclear genome of Arabidopsis and Oryza sativa were conducted. On the basis of comprehensive analysis of these results obtained, we proposed the hypothesis that organellar genes have been transferred to their nuclear genome via the process of insertion in the non-coding region of the nuclear genome, followed by obtaining the function in certain conditions and then the corresponding genes in the organellar genome disappeared. By this way of evolution, the nucleus becomes the central supremacy in the cell gradually.更多还原