三角褐指藻尿苷二磷酸葡萄糖焦磷酸化酶在碳流分配中的作用机制研究

An unicellular silica-less marine diatom, whose genome sequence has been completed, rich in EPA and other active compounds, Phaeodactylum tricornutum has very important research values in theory and application. UGP has been considered to play important roles in carbohydrate metabolism of higher plants and secondary cell wall biosynthesis, however, it is not clearly understood whether this enzyme has functions in carbon allocation and the ratio of the allocation can transfer to lipids. Previous research showed that inactivation of UGP1 in P. tricornutum led to a significant decrease in chrysolaminaran content and an increase in lipid synthesis, which suggested that UGP1 may play an important role in chrysolaminarin biosynthesis and carbon allocation. In this study, subcellualr localization of UGPs will be studied by differential centrifugation and cotransformation both UGPs and report gene in Phaeodactylum tricornutum; Substrate specificity of UGPs will be determined by prokaryotic expression; Meanwhile, to obtain different transformation algal strains, overexpression and inactivation of UGPs will be achieved in Phaeodactylum tricornutum using constructs containing enhancing promoter or inverted repeat sequences of the genes to confirm function of UGPs in carbon allocation and carbohydrate metabolism by analyzing the relationship between UGPs activity and chrysolaminaran, lipid (neutral lipid) and protein (pigment) content; Treatment to transformation algal strains using phosphate deficiency stress, low temperature and cadmium excess and so on, to clarify function of this gene in carbon allocation and chrysolaminaran metabolism, the relationship between UGP activity and chrysolaminaran, lipid (neutral lipid) and protein (pigment) content will be further analyzed; Furthermore, carbon allocation will be traced using 13C-labelled substrate (NaH13CO3) with GC–MS and LC–MS/MS.This study will contribute to lay a theoretical foundation to get good strains by gene engineering in Phaeodactylum tricornutum.

尿苷二磷酸葡萄糖焦磷酸化酶（UGP）在高等植物糖代谢中具有重要功能。三角褐指藻中有两个UGPs，申请人所在课题组研究发现，抑制UGP1基因的表达，酶活性降低，金藻昆布多糖含量减少而总脂含量增加，推测UGP1在碳流分配中发挥了重要作用，而针对UGP2还未见相关的研究。本项目拟通过报告基因共转化技术研究三角褐指藻UGPs的亚细胞定位，体外重组表达探究UGPs的底物专一性，利用高效表达载体在藻细胞内实现UGPs基因的过量表达，利用RNA干扰技术实现UGPs基因的抑制，获得不同的转基因藻株，分析转基因藻细胞中UGPs活性与金藻昆布多糖、总脂（包括中性脂）、蛋白含量变化之间的关系；以稳定同位素C13标记的NaH13CO3为唯一碳源，通过GC–MS 和 LC–MS/MS分析示踪碳流的去向，阐明UGPs在三角褐指藻金藻昆布多糖合成中的功能及碳流分配中的作用，为通过基因工程手段实现该藻的改良提供理论依据。

实现了UGP1和UGP2基因的过量表达和基因抑制，另外还实现了这两个基因的敲除（基因编辑），结果表明两个基因的表达水平与金藻昆布多糖的含量呈显著正相关，从不同角度阐明了UGPs是三角褐指藻金藻昆布多糖合成路径中的限速酶，同时发现在金藻昆布多糖合成中 UGP1 发挥的作用要大于UGP2，进一步研究发现UGP2 可能还与叶绿体中SQDG（二酰甘油硫代糖脂）的合成密切相关。 . 探究了不同转基因藻株（过表达、抑制及敲除）UGP1和UGP2活性与金藻昆布多糖、总脂、蛋白含量变化之间的关系，表明UGPs 的活性与金藻昆布多糖含量呈正相关，与总脂的含量呈负相关，对蛋白质合成的影响不显著，从不同角度明确了UGPs在三角褐指藻碳流分配中发挥重要作用。. 值得一提的是，研究过程中发现尿苷二磷酸葡萄糖（UDPG）在提高三角褐指藻细胞抗氧化能力、光合作用能力和耐高温能力方面具有重要作用，拟对其机理进行深入探究，为国家级项目申报培育了新的增长点。. 本研究为阐明硅藻金藻昆布多糖的生物合成路径提供了重要的理论依据。