类波氏真眼点藻(Eustigmatos cf. polyphem)β-1,3葡聚糖苷酶基因的功能研究

Eustigmatos cf. polyphem is a yellow-green unicellular soil microalga, which accumulates chrysolaminarin, as the primary carbon storage compound of photosynthesis.Our preliminary research showed that the chrysolaminarin content of E. cf. polyphem was gradually decreased, but the content of triacylglycerols was sharply increased under nitrogen limited conditions(N-limited). Based on the annotation of E. cf. polyphem transcriptome, we identified exo-1,3-β-glucanase (exo-Glu) and endo-1,3-β-glucanase (endo-Glu) genes involved in digesting chrysolaminarin. In this program, we will firstly construct the cDNA library of E. cf. polyphem induced by N-limited, and clone the full-length cDNA of exo-Glu and endo-Glu genes. To identify the cellular location, we will translate the fusion constructs of these genes and green fluorescent protein (GFP) in onion epidermal cells. The gene expression will be examined by real-time quantitative PCR and Northern blot. Then we will analyzed protein expression of these genes using enzyme assay, Western blot and immunocytochemistry. Based on this project, we could illuminate the detailed functional characterization of exo-Glu and endo-Glu genes in E. cf. polyphem. This research provides a substantial contribution to elucidate the relevance of chrysolaminarin metabolic pathway and triacylglycerols biosythensis.

前期研究发现，类波氏真眼点藻(E. cf. polyphem)光合固碳形成金藻昆布糖(CL，β-1,3-葡聚糖)，氮限制时细胞中CL减少，储藏性三酰甘油大量积累，测序分析证实其转录组中包含外切和内切β-1,3-葡聚糖苷酶(exo-Glu，endo-Glu)基因。氮限制如何调控exo-Glu和endo-Glu基因的表达？exo-Glu和endo-Glu在CL代谢中的作用机制是什么？这些问题的研究对于揭示微藻光合碳流分配的分子机理具有重要的理论意义。本项目拟构建该藻氮限制诱导的cDNA文库，克隆 exo-Glu和endo-Glu基因。应用GFP融合蛋白技术、荧光定量PCR、Northern blot、Western blot和免疫细胞化学等手段从亚细胞水平、转录水平和蛋白水平阐明exo-Glu和endo-Glu基因的表达模式及其在CL代谢中的重要功能，为研究微藻储藏性物质代谢转化规律奠定基础。