Assembly of Chitin-based Meshworks and their Role in Silica Morphogenesis

在这个子项目中，我们将研究几丁质网状物在生物二氧化硅形成中的作用，这是一种在硅藻中发现的不溶性有机基质。为此，我们将提取和鉴定与几丁质为基础的网络相关的蛋白质/肽。这些蛋白/肽的基因将被克隆并与标签同源表达，以研究它们在体内是否与生物硅相关。克隆6个预测的几丁质合成酶基因及其同源表达为gfp融合蛋白或肽融合蛋白，将允许在硅藻细胞中定位几丁质合成酶，以确定参与几丁质网状生物合成的酶。在同步细胞中，通过qPCR对几丁质酶基因进行转录分析，结合胞外和胞内几丁质酶的生化鉴定，有望鉴定出可能参与体内几丁质网络重塑的候选几丁质酶（在22种预测的假单胞菌几丁质酶中）。我们将尝试通过微生物几丁质酶在假单胞菌中的表达来抑制或减少几丁质网状结构的形成，并分析其对细胞生长速度和二氧化硅形态的影响。在项目后期阶段的补充研究中，我们将尝试生成和分析几丁质合成酶敲除突变体。几丁质网络与硅酸苷（高度磷酸化肽）的相互作用，以及硅酸苷在生物二氧化硅形成中的作用，将通过研究低磷酸化硅酸苷变体对体内和体内二氧化硅形成的影响来进一步研究。此外，我们将尝试产生硅酸苷敲除突变体，以分析硅酸苷对假单胞菌在体内的存活、生长和硅形态的作用。

In this subproject we will investigate the role of the chitin-based meshwork, an insoluble organic matrix found in the diatom Thalassiosira pseudonana, in biosilica formation. For this purpose we will extract and identify the proteins/peptides associated with the chitin-based meshwork. The genes of these proteins/peptides will be cloned and homologously expressed with tags to investigate whether they are associated with the biosilica in vivo. Cloning of the six predicted chitin synthase genes and their homologous expression as GFP-fusion or peptide-fusion proteins will allow for the localization of the chitin synthases in the diatom cells, to identify the enzyme(s) involved in biosynthesis of the chitin-based meshwork. Transcription analysis of chitinase genes by qPCR in synchronized cells, in combination with biochemical identification of extracellular and intracellular chitinases is expected to identify candidate chitinases (out of the 22 predicted chitinases of T. pseudonana) that may be involved in remodeling of the chitin-based meshwork in vivo. By expression of microbial chitinases in T. pseudonana we will attempt to inhibit or reduce the formation of the chitin-based meshwork, and analyze the effect on cellular growth rate and morphology of the silica. In complementary studies at later stages of the project we will attempt to generate and analyze chitin synthase knock-down mutants. The interaction of the chitin-based meshwork with silacidins (highly phosphorylated peptides), and the role of silacidins in biosilica formation will be further investigated by studying the effect of hypo-phosphorylated silacidin variants on silica formation in vivo and in vivo. Additionally, it will be attempted to generate silacidin knock-down mutants to analyze the role of silacidins on the survival, growth and silica morphology of T. pseudonana in vivo.