Expression of Vectors Transforming a Chlorella-Like Green Alga

外共生，小球藻样真核绿藻作为宿主的一组大的双链含有dna的病毒。这种藻类在有光的环境下很容易培养。这些病毒同步感染、快速复制，并最终溶解它们的藻类宿主。由于空斑测定法的发展，这是迄今为止第一个也是唯一一个利用噬菌体研究的成熟技术来研究植物病毒系统的例子。该系统的另一个好处是，病毒-藻类裂解物是细胞壁降解酶活性的丰富来源，可用于生产可引入重组DNA的原生质体。该项目的最终目标是开发一个藻类转化系统。为此，建议：(1)建立原生质体再生体系；(2)寻找来自病毒或宿主的DNA序列，这些DNA序列将赋予该藻类中质粒的自主复制；(3)确定病毒DNA复制的细胞内位点；(4)研究病毒DNA，寻找病毒基因表达调控的线索；(5)研究含有报告分子的载体，测量瞬时表达；(6)构建具有可选择标记的载体。这些目标的成功将使这一有趣的藻类系统的特征和开发成为可能，并将对植物遗传学和细胞生物学产生重大影响。

Exsymbiotic, Chlorella-like eukaryotic green algae act as hosts for a group of large double-stranded DNA-containing viruses. The algae can be readily cultured in the presence of light. The viruses synchronously infect, rapidly replicate, and ultimately lyse their algal host. As a result of the development of a plaque assay, this is the first and, thus far, only example of a plant-virus system which lends itself to investigation using established technology adapted from bacteriophage studies. An additional benefit of this system is that the viral-algal lysate is a rich source of cell wall degrading enzyme activity which can be used to produce protoplasts into which recombinant DNA may be introduced. The ultimate goal of this project is to develop an algal transformation system. Toward this end, it is proposed to: (1) develop a protoplast regeneration system for this alga; (2) search for DNA sequences from either the virus or the host which will confer autonomous replication of plasmids in this alga; (3) determine the intracellular sites of viral DNA replication; (4) study viral DNA for clues as to the regulation of viral gene expression; (5) study vectors containing reporter molecules, to measure transient expression; and (6) construct vectors with selectable markers. Success in these goals will allow the characterization and exploitation of this interesting algal system, and will have significant impact on plant genetics and cell biology.