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

在这个子项目中，我们将研究在硅藻海链藻中发现的一种不溶性有机基质--几丁质基网络在生物硅形成中的作用。为此，我们将提取和鉴定与基于几丁质的网络相关的蛋白质/肽。这些蛋白质/肽的基因将被克隆并与标签同源表达，以研究它们是否与体内生物二氧化硅相关。六个预测的几丁质合成酶基因的克隆和它们作为GFP融合蛋白或肽融合蛋白的同源表达将允许几丁质酶在硅藻细胞中的定位，以鉴定参与基于几丁质的网络的生物合成的酶。在同步化细胞中通过qPCR进行的几丁质酶基因的转录分析，结合细胞外和细胞内几丁质酶的生物化学鉴定，预期将鉴定候选几丁质酶（在T.在体内可能参与几丁质为基础的网状结构的重塑。通过在T.我们将尝试抑制或减少基于几丁质的网状结构的形成，并分析二氧化硅对细胞生长速率和形态的影响。在该项目后期的补充研究中，我们将尝试产生和分析几丁质合成酶敲低突变体。基于几丁质的网络与silacidins（高度磷酸化的肽）的相互作用，以及silacidins在生物二氧化硅形成中的作用将通过研究低磷酸化的silacidin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，我们还将尝试构建Silacidin敲低突变体，以分析Silacidin对T.体内寄生虫。

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.