枸杞蔗糖代谢关键酶基因及其调控果实糖积累的机制研究

Wolfberry ( Lycium L. ) is an important drug and food plant resources, and the fruit possess various of health functions. The traditional Chinese doctors and gourmets always attached high attention to wolfberry fruits. The sugar content and composition in the wolfberry fruits directly determined the formation of fruit qualities and synthesized some substrates of the secondary metabolites in many active ingredients(such as LBP). We previously showed that the sugar content and composition are more different in the different genotypes of wolfberry fruits. This study analyse the differences of the content and composition of LBP, LBP hydrolysis sugar in the different wolfberry fruits, and the relationship between these differences and the differences of the activities of sugar metabolism enzymes-sucrose synthase(SPS), sucrose phosphate synthase (SPS) and invertase (Inv). We can find out which of free sugars is closely associated with LBP , establish the pattern for sugar accumulation and the key enzymes for sucrose metabolism in fruits. These key enzyme genes are cloned by using RACE and set up expression characteristics in different tissues, different developmental stages and drought and saline conditions. This study analyze the relationship between these differences of sugar accumulation and the expression of the key enzyme in drought and saline conditions, reveal the molecular regulatory mechanisms in fruits sugar accumulation, which lay a foundation for improving sugar content in fruits.

枸杞（Lycium L.）是我国重要的药食两用植物资源，其果实具有多种保健功能，历来备受中外医食家高度关注。枸杞果实糖组成与含量不仅直接决定着果实品质形成，也是许多活性成分（如枸杞多糖）次生代谢产物合成的底物。申请人前期研究发现不同基因型枸杞果实糖组成与含量存在较大差异。本研究在此基础上，拟以蔗糖代谢主要酶-蔗糖合成酶（SS）、蔗糖磷酸合成酶（SPS）和蔗糖转化酶（Inv）为切入点，分析不同基因型枸杞、不同发育阶段果实中游离糖和枸杞多糖水解糖组成与含量差异，以及这些差异与SS、SPS和Inv活性差异的相关性，找出与枸杞多糖含量关联紧密的游离糖，确立枸杞果实糖积累模式及其蔗糖代谢关键酶；利用RACE技术克隆关键酶基因，研究关键酶基因在不同组织，不同发育阶段的表达特性；分析干旱盐碱条件下枸杞果实中糖积累差异与关键酶基表达的关系，揭示果实糖积累的分子调控机理，为提高枸杞果实糖积累奠定基础。

枸杞是我国重要的药食两用植物资源，其果实具有多种保健功能，历来备受中外医食家高度关注。但目前人们只注重产量，而忽视果实品质形成中有效成分积累研究。本研究以枸杞果实品质形成基础物质—糖入手，系统开展不同基因枸杞果实糖组成与含量测定，分析果实发育过程中转录组差异表达基因，研究枸杞果实发育过程中与糖代谢紧密关联的差异表达基因，筛选参与枸杞糖代谢关键基因，克隆关键基因的全长，构建关键基因高效原核表达载体，筛选出基因融合蛋白的特异性抗血清，建立基因的蛋白水平检测体系，解析关键基基因蛋白生物学特性。.重要结果：（1）搞清了不同基因型枸杞果实糖组成为果糖、葡萄糖和蔗糖，糖积累型为己糖型和果糖型；（2）利用Illumina Hiseq2500测序平台完成枸杞果实发育3个关键时期的转录组测序，共获得到87,698条unigenes；通过对三个发育时期差异表达基因比较，在显著富集的55个pathways中，筛选出33个差异表达基因，参与4个与糖代谢相关的pathways；（3）通过分析20个基因表达量与糖含量的相关性，与总糖含量显著相关基因2个，与果糖含量显著相关基因3个，与葡萄糖含量显著相关基因5个，与蔗糖含量显著相关基因5个；（4）克隆出了LbAI、LbSS、LbSPS、LbXI基因全长。（5）构建LbAI基因原核表达载体pET-28 A-LbAI-C，得到了pET- 28A-LbAI-C蛋白大小约25kD，获得了LbAI基因融合蛋白的特异性抗血清，完成枸杞果实 Western blot检测，随着果实己糖（果糖和葡萄糖）含量增加，LbAI蛋白的表达量也随之增大，验证了枸杞LbAI调控枸杞果实己糖积累关键基因。.本研究获梁希林业科学技术二等奖1项，发表论文11篇（SCI 4篇），申请发明专利3项（授权1项），出版专著1部，培养研究生2名。本研究研究为阐明枸杞品质形成的分子调控机理提供理论依据。