不同氮形态调控草莓果实糖酸代谢的分子机理研究

Nitrogen is one of most important nutrients during the growth and development of fruit trees. In our previous study, the strawberry plants were treated with diffent nitrogen forms including ammonium, nitrate, ammonium plus nitrate, and no nitrogen in sand culture experiment, and the metabolic profiling of strawberry showed the distinct cluster patterns during fruit development and maturation. The metabolism of sugars and organic acids in strawberry fruits represented the most significant changes, but the molecular mechanism is still unclear. The metabolism of sugars and organic acids in strawberry (Fragaria ananassa Duch.cv.Troyonoka) during fruit development and maturation will be investigated using untargeted (GC-MS) and targeted (HPLC) metabolic profiling technology. Data will be analyzed using a variety of chemometric analyses, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), to explore the changes of carbon-flux in strawberry fruits at different developmental stages. Meanwhile, the strawberry assembly transcriptome will be sequenced by RNA-seq, and the digital gene expression (DGE) profiling during fruit development will be analyzed using the transcriptome developed as the reference library. With the integration of metabolic profiling and DGE profiling, a detailed network within metabolites, genes, or between metabolites and genes will be constructed, which will lead to discover functional genes and their regulators closely related to the metabolism of sugars and organic acids in strawberry fruits. Newly-discovered genes (a full length of cDNA) will be cloned and their functions will be further verified in E. coli (prokaryotic) or in diploid strawberry (eukaryotic). This project will be of great significance in identifying functional genes or regulators involved in the regulation of different nitrogen forms to the metabolism of sugars and organic acids in strawberry fruits, which will facilitate enhancement of breeding germplasms and enrichment of nitrogen fertilization theory in strawberry or related fruit tree species.

氮素是果树生长发育最重要的营养元素之一。我们前期的研究发现，在草莓果实发育过程中，不同氮形态（铵态氮、硝态氮、全氮、无氮）处理果实的代谢谱存在显著差异，且引起的最显著代谢响应表现为果实糖酸代谢变化，但确切的分子机理尚不明确。本项目拟以"丰香"草莓为试材，对不同氮形态处理果实进行代谢谱分析，揭示果实发育过程中的糖酸代谢变化。同时，构建草莓转录组文库进行测序分析，并以其为参考序列，研究不同氮形态处理果实间的基因差异表达谱。整合代谢谱和基因表达谱分析结果，构建代谢物之间、基因之间、代谢物与基因之间的关联网络，筛选与果实糖酸代谢密切相关的功能基因与转录因子；对新功能基因和转录因子进行全长克隆，选用原核表达或二倍体草莓真核表达系统进行功能验证。本项目研究对于阐明不同氮形态处理调控草莓果实成熟过程中糖酸代谢的分子机理，以及丰富果树氮素营养理论、提高草莓品种改良效率，都将具有十分重要的意义。

以“丰香” 草莓为供试材料，在现花时，将供试幼苗分成四组，6次重复。采用砂培法进行不同氮形态（NO3--N、NH4+-N、NO3--N + NH4+-N、无氮）处理，花后10d开始采集幼果，至果实过熟期结束，每5d取样一次，每次取果实9-11个。.采用本课题组已建立起的草莓代谢谱分析技术平台，对供试果实样品水相提取物进行GC-MS分析。采用面积归一化法对中各色谱峰进行归一化处理，并对处理的数据进行主成分分析。多元统计分析结果显示，不同氮形态处理样品能进行较好的区分；在同一氮形态处理中，不同生育期草莓果实样品间也能进行明显区分，表明不同氮形态处理对草莓果实发育过程中代谢物形成和积累具有显著的影响。.采用SIMCA-P12.0软件对获得数据进行OPLS-DA和S-plot分析，并据此获得了与不同氮形态处理相关的生物标记物共20个，经过定性分析后，删去相同的组分进行合并，获得16个biomarker。将上述16个biomarkers映射到代谢途径中，选择登录到KEGG database中的二倍体森林草莓Fragaria vesca (fve)代谢途径作为reference pathway。结果表明，共有7个biomarkers注释到prokaryotic-type ABC transporters路径中，正好注释为Oligosaccharide, polyol, and lipid transporters和Monosaccharide transporters两类。同时，另有7个biomarkers注释到Galactose metabolism途径中，包括galactose，inositol，sorbitol，glycerol，lactose，glucose和fructose。另外，余下的包括6, 7-二羟基香豆素-β-葡萄糖苷在内的7个biomarker中，能注释到的同一个代谢途径均少于等于2个，因此，下一步研究中将不做考虑。.基于上述结果分析，不同氮形态处理主要通过半乳糖代谢途径和ABC转运蛋白，对草莓果实发育过程中糖酸代谢进行调控；但具体包括哪些功能基因？如何进行调控？我们都还不清楚！因此，我们下一步将重点围绕这些问题开展研究。