Collaborative Research: Cell Walls Resistant to Environmental Extremes: the Chlamydomonas zygaspore as a Model System

9808478 Stern Sporopollenin，通常被称为地球上最具抗性的生物聚合物，是成熟花粉粒的基本成分。 孢粉素也存在于非种子植物的孢子壁和各种微生物的休眠细胞中。 尽管这种显然是古老的化合物的重要性，但孢粉素的合成、分泌和组装途径仍然未知。 单核衣原体的接合孢子壁含有孢粉素，这种单细胞衣原体的接合孢子突变体的可用性表明C。monoica作为研究孢粉素生物合成和功能的模型。 将使用差异固定和染色，然后通过荧光或透射电子显微镜以及通过在野生型和突变株的壁形态发生期间溶解的蛋白质的电泳来分析接合孢子壁的组成。 将开发核转化程序，并使用索引粘粒文库进行转化，通过其补充突变表型的能力分离接合孢子缺陷基因的野生型等位基因。 或者，插入成熟突变体将通过核转化产生，然后分子标签将用于克隆缺陷基因。 通过这些方法克隆的基因的表达将在野生型和成熟突变体中进行比较。

9808478 Stern Sporopollenin, often referred to as the most resistant biopolymer on earth, is an essential component of mature pollen grains. Sporopollenin is also found in the spore walls of non seed plants, and in the resting cells of diverse microorganisms. Despite the importance of this apparently ancient compound, the routes of synthesis, secretion, and assembly of sporopollenin remain unknown. The zygospore wall of Chlamydomonas monoica contains sporopollenin and the availability of zygospore mutants of this unicellular alga suggests development of C. monoica as a model for the study of sporopollenin biosynthesis and function. The composition of the zygospore wall will be analyzed using differential fixation and staining followed by fluorescence or transmission electron microscopy and by electrophoresis of proteins solubilized during wall morphogenesis in wildtype and mutant strains. A nuclear transformation procedure will be developed and wildtype alleles of genes responsible for zygospore defects will be isolated by their ability to complement mutant phenotypes, using an indexed cosmid library for transformation. Alternatively, insertional maturation mutants will be generated by nuclear transformation and the molecular tag will then be used to clone the defective gene. The expression of genes cloned by these approaches will be compared in wildtype and maturation mutants.